Gallstones in a Looking-Glass: A Case Report on the Successful Laparoscopic Management of Cholelithiasis in Situs Inversus Totalis.

Situs inversus is an uncommon congenital condition where the internal organs are arranged in a mirrored or reversed orientation within the body. In this unique anatomical variation, the placement of visceral organs is flipped, presenting a mirror-image configuration relative to their standard positions. While situs inversus itself does not predispose an individual to gallbladder disorders, the anatomical variation poses unique challenges for healthcare professionals in managing abdominal pathologies. This case report describes the successful management of a 52-year-old male patient with situs inversus totalis who presented with gallstone-induced obstructive jaundice and underwent endoscopic retrograde cholangiopancreatography (ERCP) with stenting, followed by a laparoscopic cholecystectomy. The surgical procedure required exceptional visual-motor skills and extensive reorientation to accurately identify and navigate the left upper quadrant anatomy, which is the mirror image of the typical surgical approach. The case highlights the importance of thorough preoperative planning, comprehensive anatomical knowledge, and a multidisciplinary team approach to ensure favorable outcomes for patients with this rare condition.

Acute Toxicity of Hypofractionated and Conventionally Fractionated (Chemo)Radiotherapy Regimens for Bladder Cancer: An Exploratory Analysis from the RAIDER Trial.

AIMS: Adding concurrent (chemo)therapy to radiotherapy improves outcomes for muscle-invasive bladder cancer patients. A recent meta-analysis showed superior invasive locoregional disease control for a hypofractionated 55 Gy in 20 fractions schedule compared with 64 Gy in 32 fractions. In the RAIDER clinical trial, patients undergoing 20 or 32 fractions of radical radiotherapy were randomised (1:1:2) to standard radiotherapy or to standard-dose or escalated-dose adaptive radiotherapy. Neoadjuvant chemotherapy and concomitant therapy were permitted. We report exploratory analyses of acute toxicity by concomitant therapy-fractionation schedule combination. MATERIALS AND METHODS: Participants had unifocal bladder urothelial carcinoma staged T2-T4a N0 M0. Acute toxicity was assessed (Common Terminology Criteria for Adverse Events) weekly during radiotherapy and at 10 weeks after the start of treatment. Within each fractionation cohort, non-randomised comparisons of the proportion of patients reporting treatment emergent grade 2 or worse genitourinary, gastrointestinal or other adverse events at any point in the acute period were carried out using Fisher's exact tests. RESULTS: Between September 2015 and April 2020, 345 (163 receiving 20 fractions; 182 receiving 32 fractions) patients were recruited from 46 centres. The median age was 73 years; 49% received neoadjuvant chemotherapy; 71% received concomitant therapy, with 5-fluorouracil/mitomycin C most commonly used: 44/114 (39%) receiving 20 fractions; 94/130 (72%) receiving 32 fractions. The acute grade 2+ gastrointestinal toxicity rate was higher in those receiving concomitant therapy compared with radiotherapy alone in the 20-fraction cohort [54/111 (49%) versus 7/49 (14%), P < 0.001] but not in the 32-fraction cohort (P = 0.355). Grade 2+ gastrointestinal toxicity was highest for gemcitabine, with evidence of significant differences across therapies in the 32-fraction cohort (P = 0.006), with a similar pattern but no significant differences in the 20-fraction cohort (P = 0.099). There was no evidence of differences in grade 2+ genitourinary toxicity between concomitant therapies in either the 20- or 32-fraction cohorts. CONCLUSION: Grade 2+ acute adverse events are common. The toxicity profile varied by type of concomitant therapy; the gastrointestinal toxicity rate seemed to be higher in patients receiving gemcitabine.

Programming inactive RNA-binding small molecules into bioactive degraders.

Target occupancy is often insufficient to elicit biological activity, particularly for RNA, compounded by the longstanding challenges surrounding the molecular recognition of RNA structures by small molecules. Here we studied molecular recognition patterns between a natural-product-inspired small-molecule collection and three-dimensionally folded RNA structures. Mapping these interaction landscapes across the human transcriptome defined structure-activity relationships. Although RNA-binding compounds that bind to functional sites were expected to elicit a biological response, most identified interactions were predicted to be biologically inert as they bind elsewhere. We reasoned that, for such cases, an alternative strategy to modulate RNA biology is to cleave the target through a ribonuclease-targeting chimera, where an RNA-binding molecule is appended to a heterocycle that binds to and locally activates RNase L1. Overlay of the substrate specificity for RNase L with the binding landscape of small molecules revealed many favourable candidate binders that might be bioactive when converted into degraders. We provide a proof of concept, designing selective degraders for the precursor to the disease-associated microRNA-155 (pre-miR-155), JUN mRNA and MYC mRNA. Thus, small-molecule RNA-targeted degradation can be leveraged to convert strong, yet inactive, binding interactions into potent and specific modulators of RNA function.

Differences in Quality of Life and Toxicity for Male and Female Patients following Chemo(radiotherapy) for Bladder Cancer.

AIMS: BC2001, a randomised trial of treatment for muscle-invasive bladder cancer, demonstrated no difference in health-related quality of life (HRQoL) or late toxicity between patients receiving radical radiotherapy with and without chemotherapy. This secondary analysis explored sex-based differences in HRQoL and toxicity. MATERIALS AND METHODS: Participants completed the Functional Assessment of Cancer Therapy Bladder (FACT-BL) HRQoL questionnaires at baseline, end of treatment, 6 months and annually until 5 years. Clinicians assessed toxicity with the Radiation Therapy Oncology Group (RTOG) and Late Effects in Normal Tissues Subjective, Objective and Management (LENT/SOM) scoring systems at the same timepoints. The impact of sex on patient-reported HRQoL was evaluated using multivariate analyses of change in FACT-BL subscores from baseline to the timepoints of interest. For clinician-reported toxicity, differences were compared by calculating the proportion of patients with grade 3-4 toxicities occurring over the follow-up period. RESULTS: For both males and females, all FACT-BL subscores had a reduction in HRQoL at the end of treatment. For males, the mean bladder cancer subscale (BLCS) score remained stable through to year 5. For females, there was a decline in BLCS from baseline at years 2 and 3 with a return to baseline at year 5. At year 3, females had a statistically significant and clinically meaningful worsening of mean BLCS score (-5.18; 95% confidence interval -8.37 to -1.99), which was not seen in males (0.24; -0.76 to 1.23). RTOG toxicity was more frequent in females than males (27% versus 16%, P = 0.027). CONCLUSION: Results suggest that female patients treated with radiotherapy ± chemotherapy for localised bladder cancer report worse treatment-related toxicity in post-treatment years 2 and 3 than males.

Correlation of Clinician- and Patient-Reported Outcomes in the BC2001 Trial.

AIMS: To evaluate whether there is sufficient correlation between patient-reported outcomes (PROs) and clinician-reported outcomes (CROs) in bladder cancer follow-up post-radiotherapy to streamline data collection and to reduce trial follow-up burden on patients, clinicians and trial programmes. MATERIALS AND METHODS: PROs data were collected within the BC2001 trial using the Functional Assessment of Cancer Therapy specific to bladder cancer (FACT-BL) questionnaire. CROs data were collected by clinicians using Late Effects in Normal Tissues Subjective, Objective and Management (LENT/SOM). Data were collected at baseline, post-treatment, at 6 and 12 months post-randomisation and then annually to 5 years. The percentage agreement between CROs and PROs measures was evaluated at 2 and 5 years post-randomisation. Concordance was tested using the weighted Kappa statistic with 95% confidence intervals. RESULTS: Correlation was evaluated between six categories of the FACT-BL and LENT/SOM scores. At 2 years the percentage agreement across these domains ranged from 45 to 78%, with the weighted Kappa statistic between 0.07 and 0.35. Results were similar in year 5 with 48-83% agreement and kappa statistics between -0.02 and 0.21. CONCLUSION: The correlation between CROs and PROs in patients treated with radiotherapy for bladder cancer were generally poor. PROs appear to be more sensitive, with higher grade events reported. Further work is needed to evaluate whether PROs alone can be used to evaluate toxicity-related outcomes in randomised controlled trials.

Understanding the Benefit of Magnetic Resonance-guided Adaptive Radiotherapy in Rectal Cancer Patients: a Single-centre Study.

AIMS: Neoadjuvant chemoradiotherapy followed by surgery is the mainstay of treatment for patients with rectal cancer. Standard clinical target volume (CTV) to planning target volume (PTV) margins of 10 mm are used to accommodate inter- and intrafraction motion of target. Treating on magnetic resonance-integrated linear accelerators (MR-linacs) allows for online manual recontouring and adaptation (MRgART) enabling the reduction of PTV margins. The aim of this study was to investigate motion of the primary CTV (CTVA; gross tumour volume and macroscopic nodes with 10 mm expansion to cover microscopic disease) in order to develop a simultaneous integrated boost protocol for use on MR-linacs. MATERIALS AND METHODS: Patients suitable for neoadjuvant chemoradiotherapy were recruited for treatment on MR-linac using a two-phase technique; only the five phase 1 fractions on MR-linac were used for analysis. Intrafraction motion of CTVA was measured between pre-treatment and post-treatment MRI scans. In MRgART, isotropically expanded pre-treatment PTV margins from 1 to 10 mm were rigidly propagated to post-treatment MRI to determine overlap with 95% of CTVA. The PTV margin was considered acceptable if overlap was >95% in 90% of fractions. To understand the benefit of MRgART, the same methodology was repeated using a reference computed tomography planning scan for pre-treatment imaging. RESULTS: In total, nine patients were recruited between January 2018 and December 2020 with T3a-T4, N0-N2, M0 disease. Forty-five fractions were analysed in total. The median motion across all planes was 0 mm, demonstrating minimal intrafraction motion. A PTV margin of 3 and 5mm was found to be acceptable in 96 and 98% of fractions, respectively. When comparing to the computed tomography reference scan, the analysis found that PTV margins to 5 and 10 mm only acceptably covered 51 and 76% of fractions, respectively. CONCLUSION: PTV margins can be reduced to 3-5 mm in MRgART for rectal cancer treatment on MR-linac within an simultaneous integrated boost protocol.

Antibiotic resistance pattern and pathological features of avian pathogenic Escherichia coli O78:K80 in chickens.

Avian pathogenic Escherichia coli (APEC) induces colibacillosis, an acute and systemic disease, resulting in substantial economic losses in the poultry sector. This study aimed to investigate the antibiotic resistance pattern associated with frequent virulence gene distribution in APEC O78:K80 that may cause pathological alterations in chickens. The antibiogram profile showed high resistance to erythromycin, chloramphenicol, tetracycline, ampicillin, and co-trimoxazole, followed by intermediate resistance to ciprofloxacin, levofloxacin, enrofloxacin, norfloxacin, nitrofurantoin, and doxycycline hydrochloride, and sensitive to amikacin, streptomycin, gentamicin, and colistin. Virulence gene distribution identifies eight (irp-2, iutA, ompT, iss, iucD, astA, hlyF, iroN) genes through a conventional polymerase chain reaction. APEC O78:K80 caused significantly high liver enzyme concentrations, serum interleukin-6 and tumor necrosis factor-alpha levels in experimental birds. Also, infected birds have hypoproteinemia, hypoalbuminemia, and hyperglobulinemia. Necropsy examination revealed fibrinous perihepatitis and pericarditis, congested lungs, intestinal ecchymotic hemorrhages and necrotizing granulomatosis of the spleen. Histopathological examination depicted hepatocellular degeneration, myocardial necrosis, interstitial nephritis, intestinal hemorrhages and lymphopenia in the spleen. This study is the first evidence to assess the antibiotic resistance profile linked with virulence genes and clinicopathological potential of APEC O78:K80 in chickens in Pakistan, which could be a useful and rapid approach to prevent and control the disease by developing the control strategies.

Mapping Local Failure Following Bladder Radiotherapy According to Dose.

AIMS: To determine the relationship between local relapse following radical radiotherapy for muscle-invasive bladder cancer (MIBC) and radiation dose. MATERIALS AND METHODS: Patients with T2-4N0-3M0 MIBC were recruited to a phase II study assessing the feasibility of intensity-modulated radiotherapy to the bladder and pelvic lymph nodes. Patients were planned to receive 64 Gy/32 fractions to the bladder tumour, 60 Gy/32 fractions to the involved pelvic nodes and 52 Gy/32 fractions to the uninvolved bladder and pelvic nodes. Pre-treatment set-up was informed by cone-beam CT. For patients who experienced local relapse, cystoscopy and imaging (CT/MRI) was used to reconstruct the relapse gross tumour volume (GTVrelapse) on the original planning CT . GTVrelapse D98% and D95% was determined by co-registering the relapse image to the planning CT utilising deformable image registration (DIR) and rigid image registration (RIR). Failure was classified into five types based on spatial and dosimetric criteria as follows: A (central high-dose failure), B (peripheral high-dose failure), C (central elective dose failure), D (peripheral elective dose failure) and E (extraneous dose failure). RESULTS: Between June 2009 and November 2012, 38 patients were recruited. Following treatment, 18/38 (47%) patients experienced local relapse within the bladder. The median time to local relapse was 9.0 months (95% confidence interval 6.3-11.7). Seventeen of 18 patients were evaluable based on the availability of cross-sectional relapse imaging. A significant difference between DIR and RIR methods was seen. With the DIR approach, the median GTVrelapse D98% and D95% was 97% and 98% of prescribed dose, respectively. Eleven of 17 (65%) patients experienced type A failure and 6/17 (35%) patients type B failure. No patients had type C, D or E failure. MIBC failure occurred in 10/17 (59%) relapsed patients; of those, 7/11 (64%) had type A failure and 3/6 (50%) had type B failure. Non-MIBC failure occurred in 7/17 (41%) patients; 4/11 (36%) with type A failure and 3/6 (50%) with type B failure. CONCLUSION: Relapse following radiotherapy occurred within close proximity to the original bladder tumour volume and within the planned high-dose region, suggesting possible biological causes for failure. We advise caution when considering margin reduction for future reduced high-dose radiation volume or partial bladder radiotherapy protocols.

Feasibility of tumour-focused adaptive radiotherapy for bladder cancer on the MR-linac.

Bladder tumour-focused magnetic resonance image-guided adaptive radiotherapy using a 1.5 Tesla MR-linac is feasible. A full online workflow adapting to anatomy at each fraction is achievable in approximately 30 min. Intra-fraction bladder filling did not compromise target coverage with the class solution employed.

Assessing Bladder Radiotherapy Response With Quantitative Diffusion-Weighted Magnetic Resonance Imaging Analysis.

AIMS: Radiotherapy with radiosensitisation offers opportunity for cure with organ preservation in muscle-invasive bladder cancer (MIBC). Treatment response assessment and follow-up are reliant on regular endoscopic evaluation of the retained bladder. In this study we aim to determine the role of diffusion-weighted magnetic resonance imaging (DWI) and apparent diffusion coefficient (ADC) analysis to assess bladder radiotherapy response. MATERIALS AND METHODS: Patients with T2-T4aN0-3M0 MIBC suitable for radical radiotherapy were recruited prospectively to an ethics approved protocol. Following transurethral resection of the bladder tumour and prior to any treatment, magnetic resonance imaging including DWI was performed on a 1.5T system using b values of 0, 100, 150, 250, 500, 750 s/mm2. DWI was repeated 3 months after completing radiotherapy. Cystoscopy and tumour site biopsy were undertaken following this. The response was dichotomised into response (0.9, P < 0.01). ΔADCall mean of 0.16 × 10-3 mm2/s and ΔADCb100 mean 0.12 × 10-3 mm2/s predicted radiotherapy response with sensitivity/specificity/positive predictive value/negative predictive value of 92.9%/100.0%/100.0%/75.0% and 89.3%/100.0%/100.0%/66.7%, respectively. CONCLUSIONS: Quantitative DWI analysis can successfully provide non-invasive assessment of bladder radiotherapy response. Multicentre validation is required before prospective testing to inform MIBC radiotherapy follow-up schedules and decision making.

Rational Approach to Identify RNA Targets of Natural Products Enables Identification of Nocathiacin as an Inhibitor of an Oncogenic RNA.

The discovery of biofunctional natural products (NPs) has relied on the phenotypic screening of extracts and subsequent laborious work to dereplicate active NPs and define cellular targets. Herein, NPs present as crude extracts, partially purified fractions, and pure compounds were screened directly against molecular target libraries of RNA structural motifs in a library-versus-library fashion. We identified 21 hits with affinity for RNA, including one pure NP, nocathiacin I (NOC-I). The resultant data set of NOC-I-RNA fold interactions was mapped to the human transcriptome to define potential bioactive interactions. Interestingly, one of NOC-I's most preferred RNA folds is present in the nuclease processing site in the oncogenic, noncoding microRNA-18a, which NOC-I binds with low micromolar affinity. This affinity for the RNA translates into the selective inhibition of its nuclease processing in vitro and in prostate cancer cells, in which NOC-I also triggers apoptosis. In principle, adaptation of this combination of experimental and predictive approaches to dereplicate NPs from the other hits (extracts and partially purified fractions) could fundamentally transform the current paradigm and accelerate the discovery of NPs that bind RNA and their simultaneous correlation to biological targets.

Systematically Studying the Effect of Small Molecules Interacting with RNA in Cellular and Preclinical Models.

The interrogation and manipulation of biological systems by small molecules is a powerful approach in chemical biology. Ideal compounds selectively engage a target and mediate a downstream phenotypic response. Although historically small molecule drug discovery has focused on proteins and enzymes, targeting RNA is an attractive therapeutic alternative, as many disease-causing or -associated RNAs have been identified through genome-wide association studies. As the field of RNA chemical biology emerges, the systematic evaluation of target validation and modulation of target-associated pathways is of paramount importance. In this Review, through an examination of case studies, we outline the experimental characterization, including methods and tools, to evaluate comprehensively the impact of small molecules that target RNA on cellular phenotype.

Image-guided Adaptive Radiotherapy for Bladder Cancer.

Technological advancement has facilitated patient-specific radiotherapy in bladder cancer. This has been made possible by developments in image-guided radiotherapy (IGRT). Particularly transformative has been the integration of volumetric imaging into the workflow. The ability to visualise the bladder target using cone beam computed tomography and magnetic resonance imaging initially assisted with determining the magnitude of inter- and intra-fraction target change. It has led to greater confidence in ascertaining true anatomy at each fraction. The increased certainty of dose delivered to the bladder has permitted the safe reduction of planning target volume margins. IGRT has therefore improved target coverage with a reduction in integral dose to the surrounding tissue. Use of IGRT to feed back into plan and dose delivery optimisation according to the anatomy of the day has enabled adaptive radiotherapy bladder solutions. Here we undertake a review of the stepwise developments underpinning IGRT and adaptive radiotherapy strategies for external beam bladder cancer radiotherapy. We present the evidence in accordance with the framework for systematic clinical evaluation of technical innovations in radiation oncology (R-IDEAL).

The Development of Therapeutic Radiographers in Imaging and Adaptive Radiotherapy Through Clinical Trial Quality Assurance.

AIMS: Adaptive radiotherapy (ART) is an emerging advanced treatment option for bladder cancer patients. Therapeutic radiographers (RTTs) are central to the successful delivery of this treatment. The purpose of this work was to evaluate the image-guided radiotherapy (IGRT) and ART experience of RTTs before participating in the RAIDER trial. A plan of the day (PoD) quality assurance programme was then implemented. Finally, the post-trial experience of RTTs was evaluated, together with the impact of trial quality assurance participation on their routine practice. MATERIALS AND METHODS: A pre-trial questionnaire to assess the experience of the RTT staff group in IGRT and ART in bladder cancer was sent to each centre. Responses were grouped according to experience. The PoD quality assurance programme was implemented, and the RAIDER trial commenced. During stage 1 of the trial, RTTs reported difficulties in delivering PoD and the quality assurance programme was updated accordingly. A follow-up questionnaire was sent assessing experience in IGRT and ART post-trial. Any changes in routine practice were also recorded. RESULTS: The experience of RTTs in IGRT and ART pre-trial varied. For centres deemed to have RTTs with more experience, the initial PoD quality assurance programme was streamlined. For RTTs without ART experience, the full quality assurance programme was implemented, of which 508 RTTs completed. The quality assurance programme was updated (as the trial recruited) and it was mandated that at least one representative RTT (regardless of pre-trial experience) participated in the update in real-time. The purpose of the updated quality assurance programme was to provide further support to RTTs in delivering a complex treatment. Engagement with the updated quality assurance programme was high, with RTTs in 24/33 centres participating in the real-time online workshop. All 33 UK centres reported all RTTs reviewed the updated training offline. Post-trial, the RTTs' experience in IGRT and ART was increased. CONCLUSION: Overall, 508 RTTs undertook the PoD quality assurance programme. There was a high engagement of RTTs in the PoD quality assurance programme and trial. RTTs increased their experience in IGRT and ART and subsequently updated their practice for bladder cancer and other treatment sites.

Feasibility of magnetic resonance guided radiotherapy for the treatment of bladder cancer.

Whole bladder magnetic resonance image-guided radiotherapy using the 1.5 Telsa MR-linac is feasible. Full online adaptive planning workflow based on the anatomy seen at each fraction was performed. This was delivered within 45 min. Intra-fraction bladder filling did not compromise target coverage. Patients reported acceptable tolerance of treatment.

Design of a small molecule that stimulates vascular endothelial growth factor A enabled by screening RNA fold-small molecule interactions.

Vascular endothelial growth factor A (VEGFA) stimulates angiogenesis in human endothelial cells, and increasing its expression is a potential treatment for heart failure. Here, we report the design of a small molecule (TGP-377) that specifically and potently enhances VEGFA expression by the targeting of a non-coding microRNA that regulates its expression. A selection-based screen, named two-dimensional combinatorial screening, revealed preferences in small-molecule chemotypes that bind RNA and preferences in the RNA motifs that bind small molecules. The screening program increased the dataset of known RNA motif-small molecule binding partners by 20-fold. Analysis of this dataset against the RNA-mediated pathways that regulate VEGFA defined that the microRNA-377 precursor, which represses Vegfa messenger RNA translation, is druggable in a selective manner. We designed TGP-377 to potently and specifically upregulate VEGFA in human umbilical vein endothelial cells. These studies illustrate the power of two-dimensional combinatorial screening to define molecular recognition events between 'undruggable' biomolecules and small molecules, and the ability of sequence-based design to deliver efficacious structure-specific compounds.

Targeted Degradation of the Oncogenic MicroRNA 17-92 Cluster by Structure-Targeting Ligands.

Many RNAs are processed into biologically active transcripts, the aberrant expression of which can contribute to disease phenotypes. For example, the primary microRNA-17-92 (pri-miR-17-92) cluster contains six microRNAs (miRNAs) that collectively act in several disease settings. Herein, we used sequence-based design of structure-specific ligands to target a common structure in the Dicer processing sites of three miRNAs in the cluster, miR-17, miR-18a, and miR-20a, thereby inhibiting their biogenesis. The compound was optimized to afford a dimeric molecule that binds the Dicer processing site and an adjacent bulge, affording a 100-fold increase in potency. The dimer's mode of action was then extended from simple binding to direct cleavage by conjugation to bleomycin A5 in a manner that imparts RNA-selective cleavage or to indirect cleavage by recruiting an endogenous nuclease, or a ribonuclease targeting chimera (RIBOTAC). Interestingly, the dimer-bleomycin conjugate cleaves the entire pri-miR-17-92 cluster and hence functionally inhibits all six miRNAs emanating from it. The compound selectively reduced levels of the cluster in three disease models: polycystic kidney disease, prostate cancer, and breast cancer, rescuing disease-associated phenotypes in the latter two. Further, the bleomycin conjugate exerted selective effects on the miRNome and proteome in prostate cancer cells. In contrast, the RIBOTAC only depleted levels of pre- and mature miR-17, -18a, and 20a, with no effect on the primary transcript, in accordance with the cocellular localization of RNase L, the pre-miRNA targets, and the compound. These studies demonstrate a strategy to tune RNA structure-targeting compounds to the cellular localization of the target.