bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular and Microbiological Characterization of Candida auris Strains Isolated from Colonized and Infected Patients in a New York City Tertiary Care Center
Authors: Kilic Abdullah , Smithgall Marie , Shi Jun , Green Daniel , Wu Fann
Published: October 2024

Candida auris is a multidrug resistant pathogen that causes healthcare associated invasive infections and outbreaks with high mortality rates, often involving intensive care units (ICU). High rates of C. auris colonization among patients in ICUs are associated with increased risk of subsequent C. auris hospital-acquired infections. In this study, microbiological characteristics, and antifungal susceptibility patterns of C. auris isolates collected between January and December 2023 were investigated. Whole-genome sequencing was used to examine the genetic variation and molecular epidemiological characteristics. During the study period, a total of 20 patients were found to be colonized with C. auris by three site skin swab and eight of these patients subsequently developed invasive infection with C. auris. Among patients with both colonization and infection, all invasive isolates were closely related genetically to colonizing isolates and belonged to clade I. Candidemia was the most common infection identified in 4/8 (50%) patients. All isolates were resistant to fluconazole and thirteen isolates from six patients were resistant to amphotericin B. Paired isolates from patients with both colonization and invasive infection showed the same antifungal resistance patterns. All isolates had the ERG11 p.K143R mutation known to cause fluconazole resistance. Three isolates from one patient with a FKS1 hotspot region 3 p.M690I mutation were resistant to caspofungin and two isolates from one patient with a FKS1 hotspot region 2 p.R1354H mutation were resistant to caspofungin, anidulafungin and micafungin. Our study demonstrates that colonization with C. auris is associated with increased risk of infection by the same strain amongst critical ill patients. C. auris screening and implementation of adequate infection control measures are urgently needed to prevent the dissemination of C. auris infections.
Validation of Serum Tetrahydrocannabinol Assay and Its Clinical Application
Authors: Garrett Jamie , Wilson Rebecca , Laha Thomas , Liao Hsuan-Chieh (Joyce)
Published: October 2024

Background

The widespread use of marijuana across all age groups in the United States, driven by the legalization of recreational and medical cannabis, has underscored the need for accurate measurement of tetrahydrocannabinol (THC) and its metabolites in blood specimens. This requirement spans both legal and medical contexts, prompting the development of precise diagnostic tools.

Methods

We developed a high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay, utilizing a straightforward protein precipitation extraction method to quantify THC and its metabolites: THC, 11-OH-THC, THC-COOH, cannabidiol (CBD), and THC-COOH glucuronide. The assay was rigorously validated for precision, linearity, stability, carryover, and quality assurance to ensure its reliability and accuracy for clinical use.

Results

Precision studies demonstrated a coefficient of variation ranging from 1.7% to 4.9%. The analytical measurement range was established at 1-100 ng/mL for THC, CBD, and 11-OH-THC, 5-500 ng/mL for THC-COOH, and 10-1000 ng/mL for THC glucuronide, accommodating the broad spectrum of THC/metabolite concentrations observed in our patient cohort. Carryover from all analytes were negligible (<0.01%). Sample stability was confirmed for up to two weeks at -20°C and through three freeze-thaw cycles. Despite a median THC concentration of 5 ng/mL in a limited sample size (N=44), high concentrations of THC and its metabolites correlated with trauma-related incidents and a history of polysubstance abuse in emergency department admissions.

Conclusions

The LC-MS/MS assay offers superior sensitivity and specificity compared to traditional immunoassays for cannabinoid detection, effectively distinguishing between THC and its metabolites. Its implementation will offer an advanced approach for analyzing the prevalence and clinical implications of THC and metabolite levels in various patient populations, including trauma, pediatric patients, and organ transplant candidates.
Clinical Utility of Broad Range PCR for Infectious Organisms
Authors: Walley Debbie
Published: October 2024

Broad range PCR is used to identify infectious organisms when bacterial, fungal, and mycobacterial cultures are negative despite clinical evidence of infection. We performed a 6-month retrospective chart review to assess the actionability of broad range PCR and identify opportunities to improve the utility of this referral test at our institution. One hundred eleven broad range PCR tests were ordered from July to December 2023. Seventy-five (67.6%) results did not affect patient management, 18 (16.2%) results affected patient management, and the treatment impact of 18 (16.2%) results is unknown because they were from outside consult cases. There were no instances in which antibiotic therapy was completely discontinued due to a negative broad range PCR result. Concurrent in-house microbiology testing yielded a positive result in 15 (13.5%) cases, of which 8 (53%) had a negative broad range PCR result and 2 (13.3%) had a conflicting broad range PCR result. Thirty-eight (34.2%) specimens were formalin fixed paraffin embedded tissue, 29 (26.1%) were fresh tissue, 13 (11.7%) were ESWABS, and the remaining 31 specimens were various fluid types (27.9%). While fresh tissue is the preferred specimen, broad range PCR detected an organism in 20 (53%) formalin fixed paraffin embedded tissue specimens, 11 (37.9%) fresh tissue specimens, 3 (23.1%) ESWAB specimens, and 2 (6.5%) fluid specimens (abscess aspirate and cerebrospinal fluid). Improvements in physician communication and laboratory workflow are necessary to improve the specimen quality and thus the utility of this referral test at our institution.
Genotyping for Weak and Partial D Rh Status to Optimize Usage of Limited Blood Supplies
Authors: Hartsell Emily , Thomas Caleb , Beck Bethany , Patel Sachi , Barouqa Mohammad
Published: October 2024

Introduction

The Rh antigen group, expressed on erythrocytes and encoded by highly polymorphic RHD and RHCE genes on chromosome 1 (1p36-p34), is notable in transfusion medicine due to its high polymorphism, strong immunogenicity and ability to cause hemolytic transfusion reactions in patients and hemolytic disease of the fetus and newborn (HDFN). The D antigen specifically contains multiple epitopes with variations that include decreased expression (weak D) or loss of certain epitopes (partial D) which expose patients to risk of alloimmunization depending on the subtype of the Rh(D) antigen. Hence, serologic weak D testing followed by molecular testing is not performed except for donors testing negative for Rh(D) antigen and newborns typing negative for Rh(D) and born to Rh(D) negative mothers. Given the current shortages of blood supplies, understanding patients’ genotypes in the context of weak or partial D-typing and risk of alloimmunization can be essential in the management of increasingly limited supplies of red blood cell (RBC) units.

Objective

To further characterize patients that initially type negative for Rh(D) but positive for weak D with molecular testing to investigate risk of alloimmunization, retype the Rh(D) and avoid unnecessary use of Rh(D) negative RBC units during critical shortages of RBC units.

Methods

We collected the results of ABO/Rh(D) typing for patients with negative Rh(D) testing but positive weak D test over a period of four weeks in December 2023 and had D-variant molecular testing performed by a reference laboratory. Initial serologic ABO/Rh(D) was performed in tube followed by weak D testing by indirect Coombs test in tube.

Results

The total number of patients who initially tested negative for Rh(D) but positive (weak to +4) for weak-D was three patients. Two patients showed a probable genotype of RHD*01W.1 (RHD* weak type 1, 809T>G (V270G)) homozygous which indicated the presence of weak D+ type 1. Patients with weak D+ type 1 can receive Rh(D) positive red cell units, and they are not at risk of alloimmunization and developing anti-D. The third patient showed a probable genotype RHD*DVI type 2 homozygous (lack of Rh(D) exons 4 through 6) and indicated the presence of partial D antigen. Patients with partial D antigen should receive RH(D) negative red cell units as they are at risk of alloimmunization and developing anti-D.

Conclusions

Although the vast majority of Transfusion Medicine services do not perform the serologic weak D test with reflex to molecular testing, characterizing patients who test negative with initial serologic Rh(D) testing with serologic and molecular D-tests may aid in retyping the Rh(D) of patients during critical shortages, particularly for patients with ABO blood group O.
Critical values for carbon monoxide in cases admitted to the emergency department in a local hospital
Authors: Topcu Dilara Bal , Oztas Yesim Er , Kahraman Ahmet Caner
Published: October 2024

Carbon monoxide (CO) poisoning has been an important concern in winter particularly due to malfunctioning coal stoves, in many developing countries, including Turkey. Winter months, especially February, were the most dangerous periods for CO exposure. Carbon monoxide has no color or smell so has known as the silent killer. Brain, heart, and other organs are under risk in CO poisoning. Because patients usually present with non-specific symptoms, CO poisoning should be considered in the differential diagnosis of unexplained nausea and vomiting, neurological or cardiac signs and symptoms. Carboxyhemoglobin (COHb) is a reliable marker to monitor the patient. Lactate is also important as a measure of impaired systemic oxygen delivery. Fifteen patients with a diagnosis of CO poisoning were included in the study. This is the number of all cases admitted to the emergency in the last one and a half months in our local hospital located in a town with almost 48.918 residents. Blood gas analysis was performed by ABL90 FLEX, Radiometer. Reference for COHb was 0.6-1.5 % and for lactate was 0.5-1.6 mmol/L. Troponin I levels were also analyzed in some patients to assess possible myocardial ischemia. Troponin levels were analyzed by ARCHITECT i2000, Abbott and reference range was <16 ng/mL in woman, <34 ng/mL in men. Graph Pad Prism program was used for statistical analysis of the data. We had 15 cases aged between 4-56 years with COHb values with panic values (> 20%) except two of the cases with values 16.5% and 19%. The female-male ratio was 6/9. On admission to the Emergency Department, 46.6% of patients (n=7) had headache and vomiting, 40% of patients (n=6) had headache, 6.6% (n=1) fatigue and 6.6% of patients (n=1) had syncope. Median value of COHb % was 23.7(16.5-40.8), lactate was 1.9 (0.8-4.8), pH was 7.39 (7.30-7.47). Excluding three patients as outliers, lactate levels had a good correlation with carboxyhemoglobin values (R2=0.9413). It was notable that 1 patient had a higher-than-normal Troponin I level (24.9 ng/mL) who has highest lactate level (4.8 mmol/L). Four of the patients, from the same family was admitted for suspected food poisoning for the symptoms of nausea, vomiting, diarrhea. A diagnosis of CO poisoning was made after a COHb value between 16.5-25.7% for these patients. This was very critical for the accurate treatment given to those patients being hyperbaric oxygen treatment in CO poisoning cases. CO poisoning should be considered in patients admitting to the emergency department with complaints of headache, dizziness, weakness, nausea, and vomiting. Reporting panic values is a means of alerting the clinician for the right diagnosis.
Conducting Assessments of Community-Based Programs for Individuals with Dementia and their Family Caregivers
Authors: Heid Allison R , Zarit Steven H
Published: October 2024

While the gold standard for intervention science is the randomized controlled trial (RCT), there is growing emphasis on the practical implementation and evaluation of evidence-based programs into community agencies. Assessment of program efficacy by community-based organizations can confirm planned delivery of services, implementation fidelity, and demonstrate positive outcomes, which can provide justification for funding. Methods and measures used in an RCT, however, often cannot be implemented in non-research settings and different approaches are needed. This manuscript reviews the emerging literature on program evaluation work and draws upon the authors’ experiences conducting evaluations with three community-based organizations funded by Administration for Community Living to implement programs for individuals with dementia and their family care partners. This manuscript argues for an examination of the expectations of evaluation efforts for translation of research-based trials to practice and emphasizes three main strategies for assessment: (1) Development of brief tailored assessments; (2) Measuring fidelity of implementation of the program; and (3) Measuring acceptability of the program to clients.
Chevrel’s procedure for midline incisional hernia repair—not to be abandoned or forgotten
Authors: Nikolovski Andrej , Cako Dajana , Argirov Ivan , Limani Nimetula , Ulusoy Cemal
Published: October 2024

The Chevrel technique is a well-established procedure for open repair of midline incisional hernia. This retrospective single-center case series aims to present the outcome of patients with midline incisional hernia treated with a modified Chevrel technique. The modification itself comprehended the use of a single-layer continuous suture for the inverted anterior rectus abdominis muscle sheet for the creation of the “new linea alba” without overlapping. Between January 2017 and December 2023, 40 patients were operated. The overall postoperative morbidity rate was 65%. Hernia recurrence occurred in three patients (7.5%). When the basic principles of the Chevrel technique are respected and conducted, this leads to satisfactory results. The postoperative outcome of this case series showed rates of complications and recurrences in concordance with the already published literature. Therefore, this technique should always be considered for the open approach for midline incisional hernia repair.
Logics for at most countable first-order structures
Authors: Perović Aleksandar , Ognjanović Zoran , Stojanović Tatjana
Published: October 2024

In this paper we present two extensions of $\omega $-logic with infinitary inference rules, denoted $Arch$-$\omega $-logic and $non$-$Arch$-$\omega $-logic. We provide the corresponding Hilbert-style axiomatizations and prove their strong completeness with respect to countable Archimedean and non-Archimedean fields, respectively. Through several examples we illustrate a natural representation of various weight functions within the proposed framework and applications to non-monotonic reasoning and neuro-symbolic computing.
RECAPITULATING THE POST-SURGICAL BRAIN MICROENVIRONMENT OF ATYPICAL TERATOID RHABDOID TUMOURS TO IDENTIFY MEMBRANE PROTEINS FOR TARGETED THERAPY
Authors: Mistry Mr Shaylen , Porter Mr Harry , McCrorie Dr Phoebe , Snary Dr Anya , Bailey Ms Hannah , Coveney Dr Clare , Boocock Dr David , Rahman Dr Ruman , Layﬁeld Prof Robert
Published: October 2024

AIMS

Atypical Teratoid Rhabdoid Tumours (AT/RT) are rare aggressive tumours primarily arising in the hind brain of children under three, conferring median survival of <12 months. Surgery remains the only standard treatment; however tumours recur from post-surgical residual disease. Decellularised human brain cerebellum aim to recapitulate AT/RT post-surgical brain microenvironments which retain extracellular matrix (ECM) ligands, and where AT/RT plasma membrane proteins are hypothesised to represent viable molecular therapy targets.

METHOD

AT/RT, astrocyte and human brain cerebellum (decellularised and non-decellularised) membrane protein fractions were extracted and analysed using liquid chromatography-mass spectrometry. Proteomic analysis tools were integrated to identify differential expressed proteins and identify shared correlations.

RESULTS

Decellularised cerebellar ECM exhibited an enriched membrane proteome relative to total proteome, evidence of complete removal of intracellular components. Furthermore, decellularised and non-decellularised cerebellar tissue expressed comparable levels of membrane proteins and protein networks, indicative of ECM ligand retention. AT/RT and astrocyte KEGG Pathway analysis revealed speciﬁc oncology-based pathway expression in membrane protein compared to total protein, reinforcing membrane proteins as more visible therapeutic targets. Between the top 20 expressed proteins, ATP1A1 was the only membrane protein shared exclusively between all AT/RT cells and astrocytes, suggesting a critical functional role. Membrane proteins showing shared expression when normalised against each AT/RT cell line included ATP1A1, HSPD1, GNA13 and SLC3A2, indicating biological similarities between AT/RT molecular subtypes, which may offer candidate ubiquitous therapy targets. Membrane proteins expressed similarly in AT/RT-MYC molecular subtypes include ATP1A1, SLC7A5 and EEF1A1. STRING membrane pathway analysis also identiﬁed shared proteins between AT/RT and astrocyte cell populations.

CONCLUSION

AT/RT membrane protein pathways provide ideal therapeutic targets directly amenable for drug repurposing. Future work prioritises proteomic analysis utilising 3D AT/RT spheroids and cerebellum brain tissue to maximise accurately recapitulating the AT/RT post-surgical microenvironment. Cerebellum decellularisation still permits retention of essential proteins and ligands.
MAPPING GLIOMAS: A RADIOGRAPHIC AND SPATIAL ANALYSIS ACCORDING TO THE 2021 WHO CLASSIﬁCATION
Authors: Li Mr Yonghao , Wan Mr Yizhou , Price Prof Stephen
Published: October 2024

AIMS

The spatial positioning of gliomas within the brain signiﬁcantly inﬂuences prognosis, potentially mirroring the genetic landscape of their precursor cells. Furthermore, the association between glioma subtypes and their spatial distribution underscores a complex interplay that may illuminate underlying pathogenetic mechanisms. This study explores the intricate relationship between the location of gliomas and their classiﬁcation according to the 2021 WHO guidelines, aiming to unravel the spatial and genetic nuances that deﬁne glioma heterogeneity.

METHOD

In this study, preoperative anatomical MRI data from 242 glioma patients were meticulously analyzed to elucidate spatial distribution patterns in relation to tumor genetics. The imaging data underwent registration to the MNI-152 standard brain space, facilitating uniform analysis. Subsequent tumor segmentation and division of the brain into 84 distinct subregions allowed for a detailed examination of tumor locations. A statistical analysis was conducted to explore the stereospeciﬁc frequency of glioma occurrence across these subregions, correlating ﬁndings with tumor grade and pivotal genetic markers, including MGMT promoter methylation, IDH1 mutation, 1p/19q co-deletion, and CDKN2A/B homozygous deletion.

RESULTS

We developed 21 distinct tumor frequency atlases based on subtypes (Glioblastoma, Oligodendroglioma, Astrocytoma), biomarker status (IDH, 1p19q, MGMT, CDKN2A/B), and grade (2, 3, 4). Employing probability vectors for statistical analysis revealed a signiﬁcant correlation among different subtypes, locations and overall survival. Furthermore, we used a Visualization tool to build a 3D heatmap for each tumor subtype, more intuitively illustrating spatial correlations.

CONCLUSION

Our study underscores the prognostic value of tumor location and its correlation with glioma subtypes and patient outcomes, suggesting a potential pathway to personalized treatments. Future efforts will focus on incorporating multi-omics data into spatial radiographic analyses.
GLIOMA MONITORING VIA LONGITUDINAL INTRACRANIAL CSF CELL-FREE DNA: ONE PATIENT AT A TIME
Authors: Riviere-Cazaux Ms Cecile , Dong Dr Xiaoxi , Mo Dr Wei , Dai Dr Chao , Campian Dr Jian , Ruff Dr Michael , Eckel-Passow Dr Jeanette , Kaufmann Dr Timothy , Kizilbash Dr Sani , Warrington Dr Arthur , Jenkins Dr Robert , Du Dr Pan , Jia Dr Shidong , Burns Dr Terry
Published: October 2024

AIMS

Methods for disease monitoring in glioma are currently limited. Liquid biopsies are of increasing interest within the ﬁeld but have been challenging due to the blood-brain barrier. To bypass this challenge, we aimed to assess the technical and clinical feasibility of obtaining intracranial cerebrospinal ﬂuid (CSF) for disease monitoring via cell-free DNA (cfDNA).

METHOD

Adults with gliomas were recruited for longitudinal intracranial CSF collection via CSF access devices, including Ommaya reservoirs in four patients and an existing VP shunt in one patient. cfDNA was then extracted and analyzed using two sequencing platforms, PredicineCARE for cancer variant proﬁling and PredicineSCORE for low-pass whole genome sequencing (LP-WGS). This analysis generated genomic aberration proﬁles and genome wide copy number burden (CNB) score.

RESULTS

Across ﬁve patients, thirty-ﬁve CSF samples were obtained (median volume: 3.80 mL; 0.5-5 mL), with 30 samples (85.7%) yielding suﬃcient cfDNA. Tumor fraction increased by 6.28x and 2.87x in two patients with radiographic disease progression and decreased by 0.08x and 0.04x in another two after chemoradiation. In another patient with pseudoprogression, tumor fraction decreased from 0.26 after resection to unmeasurable levels. Patient speciﬁc tumor-associated variant allelic frequencies (VAFs), including TP53, PTEN, and TERT, decreased within individual patients after resection and chemoradiation. In two patients with IDH-mutant gliomas, IDH1 VAF after resection correlated with decreased CSF D-2-hydroxyglutarate (D-2-HG) levels (0.64x and 0.62x, respectively, for the ﬁrst patient, and 0.01x and 0.07x for the other patient). Both IDH1 VAF and D-2-HG increased in a patient with radiographic progression (2.56x and 9.21x, respectively). CNB decreased below the limit of quantiﬁcation during treatment and increased above the limit at progression.

CONCLUSION

Longitudinal CSF cfDNA can feasibly be obtained via CSF access devices in patients with gliomas during their disease course. Further sample collection and analysis are ongoing to determine the thresholds of change indicative of treatment response and progression.
LOCATE-SM PROTOCOL: LOCATION ORIENTED CORTICAL AND SUB-CORTICAL ASSESSMENT FOR THERAPEUTIC EVALUATION- SENSORY-MOTOR TESTING IN AWAKE CRANIOTOMY
Authors: Stephenson Mrs Elisa , Cooper Mrs Sara , Ewan Ms Seonaid-Mairi , Bolton Mr William Stephen , Mathew Mr Ryan
Published: October 2024

AIMS

Awake craniotomy is a well-established surgical procedure to debulk intracranial tumours in eloquent areas (‘functional hubs’). Intraoperative mapping facilitates maximal safe debulking. Review of current sensory motor assessment tools highlighted the absence of a comprehensive ‘location to function’ protocol. We aimed to develop a new protocol that enables ‘location to function’ sensory-motor task selection in preparation for, and during, awake craniotomy.

METHOD

A literature review demonstrated multiple different intraoperative strategies exist for the preservation of sensory-motor function in awake craniotomy. Incorporating these with functional neuroanatomy, and consensus agreement in the Tessa Jowell Academy Physiotherapy Awake Craniotomy Working Group and the National Brain Mapping MDT, led to the development of testing strategies aligned with ‘location to function’ task selection. Testing strategies were peer-reviewed by three specialist Neuro-Oncology physiotherapists.

RESULTS

A protocol (LOCATE-SM) has been developed at Leeds to identify critical cortical and subcortical sensory-motor functions that are aligned with anatomo-functional models. Physiotherapists have used this to select individualised pre-, intra- and postoperative tests depending on tumour location and sensory-motor impairment. Additionally, this facilitates intraoperative mapping of eloquent functional regions that are variably located. Cases using LOCATE-SM are presented.

CONCLUSION

LOCATE-SM is a new awake sensory-motor testing protocol developed by specialised Neuro-Oncology physiotherapists at a Tessa Jowell Centre of Excellence (Leeds). Current cases demonstrate that it leads to greater consistency between physiotherapists and speciﬁcity in ‘location to function’ sensory-motor task selection. Future work will systematically analyse the outcomes (onco-functional balance) of using this protocol, and validation in other centres.
PRECISE-GBM: A RADIOGENOMIC MACHINE LEARNING BASED STUDY TO IDENTIFY PREDICTIVE RADIOMICS FOR EVALUATION OF CANCER IMMUNE SIGNATURE IN IDHW GLIOBLASTOMA
Authors: Ghimire Dr Prajwal , Modat Dr Marc , Booth Dr Thomas
Published: October 2024

AIMS

The aim of the study is to identify machine learning model based on radiological biomarkers that can predict immune status within tumor microenvironment of IDH wildtype glioblastoma.

METHOD

This is a retrospective multicenter machine learning based study utilizing open access anonymized matched radiogenomic data of TCGA-GBM, CPTAC, Ivy GAP and REMBRANDT datasets. REMBRANDT data acted as hold out dataset. Imaging data consisted of MRI based radiomics features extracted from deep learning based segmented tumors and transcriptomic (RNA seq/microarray) data consisted of immune scores extracted using CIBERSORTx. Outliers were identiﬁed and ﬁltered using principal component analysis. Combat and neuroCombat were utilized for data harmonization for each data type respectively. Radiomic feature selection was performed using LASSO technique with cross validation. Support vector machine (SVM) was trained and tested with 80:20 radiomics data split and a median based binary label of immune score (low and high). Validation of the trained model was performed with hold out dataset. Data query and analysis was performed using python and R statistical software.

RESULTS

One hundred one patients were included in the study with 15 being on the hold out set. Trained SVM model predicted the immune score label with AUC of 0.79 and balanced accuracy of 0.76 (recall 0.75, F1 score 0.75, precision 0.75) in the test data. In the holdout set, the model predicted the immune score with AUC of 0.70 with speciﬁcity of 75% in predicting the cold immune cases. The radiomic signature that acted as the label for this model were a group of ﬁrst order and second order radiomic features.

CONCLUSION

Radiogenomic SVM model non-invasively predicts immune status in wildtype glioblastoma microenvironment with good accuracy. This model has potential to be utilized in a prospective glioblastoma clinical trial for immunotherapy.
INVESTIGATING GLIOBLASTOMA STEM CELL DIFFERENTIATION IN HUMAN BRAIN 3D ORGANOID CO-CULTURES
Authors: Azzarelli Dr Roberta , Morcom Dr Laura , Rowitch Prof David , Philpott Prof Anna
Published: October 2024

AIMS

Glioblastoma (GBM) recurrence is rooted in the ability of residual stem cells to survive treatment and drive relapse. One way to eradicate glioblastoma stem cells (GSCs) is to differentiate them into post-mitotic, non-tumorigenic cell types. Here we aim to differentiate GSCs via genetic manipulation, and test how their response differs in 2D and 3D cultures.

METHOD

We generated a co-culture model to grow GSC lines with iPSC-derived 3D human brain organoids. Before co-culture, the GSC lines were genetically engineered to overexpress a Doxycycline (Dox) inducible form of the proneural transcription factor ASCL1 (wild type and phosphorylation defective). Live imaging and immunochemistry were performed to analyse tumor composition.

RESULTS

Our data show that activation of the proneural factor ASCL1 in GSCs when co-cultured with 3D human brain organoids drives reduction in tumor growth and that inhibition of ASCL1 phosphorylation only slightly improves this effect. In comparison to data in 2D cultures, the effect of ASCL1 activation seems enhanced in 3D organoids. At day 7 and 14 of ASCL1 induction, there are only few cancer cells left in the organoids, while, in 2D, wildtype ASCL1-expressing cells continue to grow, even if at a slower rate than control GSCs.

CONCLUSION

Our data show that cancer stem cells embedded in 3D human brain organoids behave differently than when in 2D culture on plastic and respond more effectively to differentiation treatments. Our model provides a platform to test novel treatments for GBM.
MODELLING GBM RECURRENCE: THE INﬂUENCE OF PERI-LESIONAL OEDEMA AND THE DISCONNECTOME
Authors: Tariq Ms Manaal , Ruﬄe Dr James , Mohinta Ms Samia , Brothwell Dr Morag , Brandner Prof Sebastian , Nachev Prof Parashkev , Hyare Harpreet
Published: October 2024

AIMS

Residual tumour burden after surgery in GBM patients is a prognostic imaging biomarker, often involving the area of T2 signal elevation surrounding the tumour core, histopathologically representing inﬁltrative oedema with tumour cells. We present the ﬁrst stage of a model to improve the prediction GBM recurrence by speciﬁcally investigating the peri-lesional oedema compartment.

METHOD

UK-wide sample of histologically proven IDHwt GBMs with MRIs at the time of diagnosis underwent state-of- the-art in-house tumour segmentation pipeline and radiomic feature extraction of whole-tumour, enhancing, non-enhancing and oedema components, and masked disconnectome map components. Of these, early GBM recurrence, within 6 months after completion of radiotherapy, was curated and distribution mapped across the brain.

RESULTS

1464 patients were identiﬁed: male = 901, median age 63 years, average overall survival 597 days. The radiomic features: Compactness 1 and 2, Surface Area, Spherical Disproportion, Sphericity, and Maximum 2D Diameter column of the disconnectome-related perilesional oedema compartment were most predictive of survival outcome (p value <0.01). Of these, 47 patients with average overall survival 357 days and average time from end of radiotherapy to progression of 131 days were identiﬁed.

CONCLUSION

Our early results reveal the relationship between perilesional oedema MRI features and survival, with disconnectome-related perilesional oedema features most predictive. This suggests that extended brain network aberrations caused by gliomas may also inﬂuence tumour recurrence. Further work will focus on the development of more robust predictive models of tumour recurrence that have potential for signiﬁcant impact on surgical and radiotherapy planning.
FROM VEIN TO BRAIN: ENHANCING TRAﬃCKING OF CAR T-CELLS IN DIFFUSE MIDLINE GLIOMA
Authors: Saukila Dr Louise Steele , Hotblack Dr Alastair , Burke Dr Christina , Rodriguez Ms Carmen , Righi Dr Matteo , Kalber Dr Tammy , De-Cola Dr Antonia , McNicholas Mr Michael , Pathania Dr Manav , Pule Dr Martin , Straathof Dr Karin
Published: October 2024

AIMS

H3K27M-altered diffuse midline glioma (DMG) is the deadliest childhood brain tumour, with treatment limited by anatomical location and chemoresistance; palliative radiotherapy remains the mainstay of treatment. Chimeric antigen receptor (CAR) T-cell therapy produces promising pre-clinical results in treating these tumours whilst leaving interspersed normal brain cells intact. Early clinical outcomes of GD2-CAR T-cell therapy for H3K27M-altered DMG demonstrate short-lived neurological and radiological improvement. Enhancing traﬃcking to and inﬁltration into the tumour core may enhance CAR T-cell eﬃcacy. Here, advanced T-cell engineering modules are tested for their ability to improve CAR T-cell traﬃcking.

METHOD

A library of T-cell engineering modules was created each with a unique DNA barcode to allow identiﬁcation of CAR T-cells expressing a given module. The library includes chemokine receptors (CCR) to improve tumour homing by chemotaxis towards tumour-secreted chemokines, adhesion molecules to increase extravasation across the blood-brain-barrier, and molecules to degrade upregulated tumour extracellular matrix (ECM) components to improve CAR T-cell motility through the tumour. Murine T-cells were co-transduced with a CAR and barcoded advanced T-cell engineering modules and administrated intravenously in syngeneic models of high-grade glioma (HGG)/DMG. Tumour, normal brain, bone marrow, spleen and liver tissue were collected at several time points post CAR T-cell administration to assess presence of CAR T-cells using genetic barcodes.

RESULTS

Co-expression of advanced T-cell engineering modules did not impair CAR T-cell function in vitro. CARs co-expressing selected CCRs were preferentially detected in tumour tissue in mice with GL261-EGFRvIII or H3.3K27Mp53LOFPDGFRAWT-engrafted tumours. Findings were further validated by ﬂow cytometry detection of selected CCR-expressing CAR T-cells as compared to CAR T-cells without additional engineering components. Similar experiments for adhesion molecules and ECM modiﬁers are planned.

CONCLUSION

Results to date indicate that CAR T-cells co-expressing selected CCRs enhance their homing to HGG/DMG tumours, encouraging further in vivo research to assess impact on CAR T-cell eﬃcacy.
CHARACTERISATION OF THE RESPONSE OF PRE-CLINICAL MODELS OF GLIOMA TO METABOLIC THERAPIES USING IN VIVO MAGNETIC RESONANCE SPECTROSCOPY
Authors: Perryman Dr Richard , Baxan Dr Nicoleta , Syed Dr Nelofer
Published: October 2024

AIMS

Magnetic resonance imaging (MRI) is routinely used to diagnose and monitor brain tumours. Magnetic resonance spectroscopy (MRS) allows us to record the metabolic proﬁle of tumours in situ. This provides a non-invasive method to monitor the response of tumours to metabolic therapies. We aimed to characterise the response of pre-clinical models of glioma to metabolic therapies such as arginine deprivation and the ketogenic diet (KD) by MRS, to better understand what metabolic changes are occurring in the tumour.

METHOD

We performed orthotopic injection of three different cell lines, including the syngeneic models GL261 and CT2A, as well as a primary GBM cell line, into the striatum of C57BL/6 and athymic nude mice respectively. Mice were randomised to either arginine deprivation treatment using pegylated arginine deiminase (ADI), or KD, plus controls. In each animal, we collected single voxel MRS data using STEAM on both the tumour itself, and healthy brain. MRS data was exported in jMRUI format and analysed in R using the spant package, followed by PCA and OPLS-DA.

RESULTS

PCA showed that there is clear distinction between healthy and tumour spectra across all models. OPLS-DA indicated that the lipid and choline spectral regions were signiﬁcantly associated with tumour, whereas the NAA and glutamine/glutamate regions were signiﬁcantly associated with healthy brain. Integration of speciﬁc peaks associated with these metabolites conﬁrmed these results. Subset analysis indicated distinct metabolic signatures between the syngeneic models and the primary GBM model. Furthermore, there is evidence to suggest that tumours in animals treated with arginine deprivation have a unique metabolic signature.

CONCLUSION

This data indicates that the tumour speciﬁc response to metabolic therapies can be measured by routine MRS screening. This would allow a non-invasive method by which clinicians could monitor the effectiveness of metabolic therapies in patients with gliomas.
APPLICATION OF IN VITRO MODELS: INCORPORATING BLOOD BRAIN BARRIER MODELS WITH GLIOMA, AND THE USE OF MICROﬂUIDICS
Authors: Taylor-Chilton Ms Eleanor , Baldwin Dr Lydia , Stead Dr Lucy , Greenman Prof John , Wade Dr Mark , Rabie Dr Tamer , Alder Dr Jane
Published: October 2024

AIMS

The blood brain barrier (BBB) presents one of the main obstacles for the development of novel glioblastoma treatments. The various transporters and enzymes within the BBB reduce the disposition of novel therapies into the brain. There is a need for an in vitro BBB-glioma model with proven eﬃcacy of predicting CNS delivery at earlier stages in the drug discovery pipeline.

METHOD

A BBB-glioma lab-on-a-chip model could enable rapid identiﬁcation of novel therapies that can cross the BBB at earlier stages of the drug development process, before going to clinical trials. The application of microﬂuidic technology allows for the detection of soluble tissue-derived factors and the assessment of the levels of soluble markers released from malignant tissue. Adding ‘ﬂow’ to the model, is more reﬂective of the dynamic ﬂow present at the BBB.

RESULTS

Validation studies of the current BBB model and converting this model over to a lab-on-a-chip model have shown the presence of an endothelial cell barrier and the tight junction proteins Zo-1 and Claudin-1, through western blotting, immunocytochemistry, and permeability experiments using varying molecular weights of FitC-dextran. These validation studies have been completed using brain endothelial monolayers on polycarbonate transwell inserts, with the immortalised cell line HCMEC/d3, and the short-term culture cells HBMEC. Also, validation studies using a co-culture of endothelial cells, astrocytes and pericytes have begun, using the same techniques to show a barrier has formed, on both the current BBB model and the lab-on-a-chip model.

CONCLUSION

The aim of this study is to combine the use of an all-human BBB-glioma model with lab-on-a-chip technology to produce a high throughput model to test novel therapies at an earlier stage. The lab-on-a-chip model will be validated for the purpose of measuring permeability and drug response and to investigate the expression of novel targets verses expression in conventional models.
TARGETING RESIDUAL CANCER FROM THE INVASIVE MARGIN OF GLIOBLASTOMA USING COMPUTATIONAL TOOLKITS
Authors: Shah Ms Maria , Smith Dr Stuart , Bakker Dr Emyr , Rahman Dr Ruman
Published: October 2024

AIMS

Inﬁltrative margin GBM, as a proxy for residual disease post-surgery, can be identiﬁed by 5-aminolevulininc acid (5ALA) to facilitate the discovery of uniquely expressed or upregulated molecular pathways. Complementary bioinformatics approaches offer reproducible ways to enable interoperability of heterogenous data sources towards identiﬁcation of therapeutic targets associated with GBM inﬁltration.

METHOD

RNA-seq primary tumour data was ﬁrst categorised as: 5ALA+ (inﬁltrative margin) vs tumour core and 5ALA+ vs 5ALA- (reactive brain). Two levels of ﬁltration (genes with adjusted p-value <0.05; degree of fold change with log2>1.5) were applied to genes, creating two separate gene lists. Cross-referencing gene lists identiﬁed transcripts upregulated in both 5ALA+ vs core and 5ALA+ vs 5ALA-. This candidate gene list was inputted into STRING to generate an uncurated protein-protein interaction (PPI) network, which returned a signiﬁcant PPI enrichment p-value to conﬁrm network connectivity, and next inputted into Metascape to elucidate biological processes associated with tumour inﬁltration.

RESULTS

Of 141 upregulated 5ALA+ genes TCP10L3, HELT, MGAM, ALOX5 and FOXI1 exhibited the greatest fold change. The most prevalent 5ALA+ biological processes were primitive streak formation, disaccharide metabolic process and sodium ion transmembrane transport. LOC157273, LINC00200, LINC00656, LOC285626 and LINC01568 were identiﬁed as long non-coding RNAs (lncRNA) most abundant in the inﬁltrative margin of GBM relative to tumour core and reactive brain. Regulatory associations between candidate 5ALA+ lncRNA and genes, and in silico drug repurposing via molecular docking, are being investigated.

CONCLUSION

The plasticity of GBM warrants analysis of the GBM inﬁltrative margin via computational toolkits to enhance understanding of upregulated molecular pathways, functionally associated with an invasive phenotype.
AN EX-VIVO SPECTRAL AND LIFETIME-DECAY ANALYSIS OF 5-ALA DERIVED PPIX ﬂUORESCENCE: OBJECTIVE ﬂUORESCENCE MAPPING IN GLIOMA SURGERY
Authors: Elliot Mr Matthew , Segaud Dr Silvere , MacCormac Mr Oscar , Patel Ms Sabina , Reisz Dr Zita , Lavrador Mr Jose , Vergani Mr Francesco , Bhangoo Mr Ranjeev , Ashkan Prof Keyoumars , Al-Sarraj Prof Safa , Stasiuk Dr Graeme , Xie Dr Yijing , Vercauteren Prof Tom , Shapey Mr Jonathan
Published: October 2024

AIMS

5ALA/Protoporphyrin IX (PpIX) ﬂuorescence guided surgery (FGS) in high-grade glioma has been shown to increase resection and survival, despite this beneﬁts are limited in low-grade glioma by low emission and subjective interpretation. Quantitative ﬂuorescence could address these issues, however is challenging due to photobleaching and variations in tissue induced distortion and PpIX photochemical state that are poorly understood. Lifetime-decay measures intrinsic ﬂuorophore properties and is not inﬂuenced by these factors, but is diﬃcult to measure intra-operatively. We used paired optical and ﬂuorescence spectroscopy with lifetime decay mapping to characterise PpIX emission within the glioma tissue microenvironment.

METHOD

Biopsies were collected from 20 patients undergoing glioma FGS, along with 4 controls from partial temporal lobectomies. Paired measurements of emission and lifetime were matched with optical property readings. Lifetime and emission ratios at 635 and 620nm were calculated (PpIX635/620), corresponding to the primary emission peak for the photochemical states of PpIX in tissue. Raw and optically corrected emission was correlated with lifetime decay.

RESULTS

205 specimens were analysed by WHO grade (56 Grade4, 66 Grade3, 37 Grade2, 20 control). PpIX635/620 emission was 0.93 in control (CI 0.91-0.95), 1.03 in grade 2 tumours (CI 0.95-1.26), 1.56 in grade 3 (CI 1.22-1.91) and 6.99 (CI 5.78-8.20) in grade 4. PpIX635/620 lifetime correlated with emission (R2 =0.98, range 0.94-1.57). Correlation of emission with lifetime readings improved signiﬁcantly following optical correction at 635nm (raw R2 =0.81, corrected R2 =0.90) and 620nm (raw R2 =0.44, corrected R2 =0.86).

CONCLUSION

PpIX620 and PpIX635 photochemical states are present in human glioma, with an increase in PpIX635/620 suggesting increased WHO grade. The ability to distinguish these forms holds the potential to provide real-time data for tumour differentiation and pathology. Accurate characterisation and correction of tissue induced distortion signiﬁcantly improves the ability to detect, characterise and quantify PpIX intra-operatively.
PATTERNS OF RADIOTHERAPY PRACTICE FOR FUNCTIONING PITUITARY TUMOURS - A SURVEY OF UK CENTRES
Authors: Pilar Dr Avinash , Ronneberger Dr Ruth , Tyson Ms Charlotte , Baldeweg Prof Stephanie , Kosmin Dr Michael
Published: October 2024

AIMS

Pituitary adenomas are benign tumours that are classiﬁed as functioning or non-functioning based on the production of excess pituitary hormones. Functioning adenomas (FA) typically present as hypersecretory syndromes including acromegaly and Cushing’s disease. In FA, radiotherapy is used when surgery fails to achieve hormone normalisation and/or when medical treatment does not control hormone excess. In the absence of prospective studies that clearly deﬁne the optimal radiotherapy technique in FA, it is expected that there will be variability in practice. A survey of UK neuro-oncology centres was undertaken, aiming to establish the extent of this variability in FA.

METHOD

A survey was distributed electronically to 20 neuro-oncology departments across the UK. Data were collected on patient numbers, radiotherapy indications, techniques, dosing, and target volume delineation.

RESULTS

Responses were received from eight centres (40% response rate), treating a total of approximately 32 FA per annum. There was variability in techniques employed to treat FA. While four centres treated most FA with IMRT, two treat most with stereotactic radiosurgery (SRS), and two with fractionated stereotactic radiotherapy (fSRT). The most common dose used for IMRT treatment was 45Gy in 25 fractions (n=7), with 50.4Gy in 28 fractions, 50Gy in 30 fractions and 54Gy in 30 fractions also reported. Use of margins around residual disease (n=5) and inclusion of involved cavernous sinuses and surgical tract (n=3) within clinical target volumes also varied. Most centres (n=7) would agree to a change in target volume delineation practices for the purpose of a clinical trial in FA.

CONCLUSION

This survey found that there is variability in radiotherapy dosing, technique, and target volume delineation practices for radiotherapy to FA across the UK. All centres but one would accept changes in their practice within the context of a prospective clinical trial of radiotherapy for FA.
NEURONAL INJURY DRIVES GLIOBLASTOMA INITIATION
Authors: Baronik Mr Zan Florjanic , Clements Dr Melanie , Tang Dr Wenhao , Ragdale Dr Holly Simpson , Lindsay Ms Rachel , Marguerat Dr Sam , Hill Mr Ciaran , Parrinello Prof Simona
Published: October 2024

AIMS

Glioblastoma (GBM), the most common primary brain malignancy in adults, remains incurable. Pervasive heterogeneity and plasticity underpin therapy resistance and lethal recurrence, suggesting that prevention or earlier intervention may be required. Improved understanding of GBM initiation is a prerequisite for achieving this goal. GBM frequently originates from neural stem cells (NSCs) of the subventricular zone, which, upon acquisition of mutations, migrate away from their niche and form tumours at distal sites through unknown mechanisms. Here, we explored GBM initiation and its microenvironment.

METHOD

We combined a murine somatic GBM model which faithfully recapitulates the human disease with PDX models and patient tissue datasets. We used immunohistochemistry, scRNA-sequencing, immuno-phenotyping, behavioural and survival studies and in vivo gain- and loss-of-function experiments in Sarm1-/- lines.

RESULTS

Time-course analysis of tumour development identiﬁed neuronal degeneration as an early event in GBM tumourigenesis, occurring preferentially in white matter, where we ﬁnd mutated NSCs are immediately rerouted. Axonal loss was accompanied by a robust inﬂammatory response, marked by microglial and astrocytic activation. Neuronal degeneration played a causative role, in that transection injury of corpus callosum axons accelerated tumourigenesis and, conversely, GBM development was delayed in Sarm1-/- mice with impaired Wallerian degeneration, a major mode of neuronal death following injury. Consistently, terminal Sarm1-/- tumours were more diffuse, contained more immature tumour cells and were less immune-inﬁltrated than their WT counterparts. Strikingly, behavioural studies revealed that Sarm1-/- tumour-bearing mice also retained improved motor performance compared to WT mice, even at terminal disease stages.

CONCLUSION

Our work provides important insights into GBM development, identifying white matter injury as a key initiating event, elicited by early tumour cells and orchestrated by neuronal degeneration. It identiﬁes Wallerian degeneration, a druggable pathway already in clinical trials for neurodegenerative diseases, as a therapeutic target for GBM, with the potential to extend survival and ameliorate cognition.