Long-term persistence and functionality of adoptively transferred antigen-specific T cells with genetically ablated PD-1 expression.

Engagement of the inhibitory T cell receptor programmed cell death protein 1 (PD-1) associates with dysfunctional states of pathogen- or tumor-specific T cells. Accordingly, systemic antibody-mediated blockade of PD-1 has become a central target for immunotherapies but is also associated with severe toxicities due to loss of peripheral tolerance. Therefore, selective ablation of PD-1 expression on adoptively transferred T cells through direct genetic knockout (KO) is currently being explored as an alternative therapeutic approach. However, since PD-1 might also be required for the regulation of physiological T cell function and differentiation, the suitability of PD-1 as an engineering target is controversial. In this study, we systematically investigated the maintenance of T cell functionality after CRISPR/Cas9-mediated PD-1 KO in vivo during and after acute and chronic antigen encounter. Under all tested conditions, PD-1 ablation preserved the persistence, differentiation, and memory formation of adoptively transferred receptor transgenic T cells. Functional PD-1 KO T cells expressing chimeric antigen receptors (CARs) targeting CD19 could be robustly detected for over 390 d in a syngeneic immunocompetent mouse model, in which constant antigen exposure was provided by continuous B cell renewal, representing the longest in vivo follow-up of CAR-T cells described to date. PD-1 KO CAR-T cells showed no evidence for malignant transformation during the entire observation period. Our data demonstrate that genetic ablation of PD-1 does not impair functionality and longevity of adoptively transferred T cells per se and therefore may be pursued more generally in engineered T cell-based immunotherapy to overcome a central immunosuppressive axis.

Paediatric Free Open Access Meducation (FOAM): behaviours, trends and implications.

INTRODUCTION: Free Open Access Meducation (FOAM) describes online resources assisting learning in medicine. Little is known about users or their behaviours. METHODS: Using Google Analytics for a popular FOAM site (www.paedatricfoam.com), we explored user demographics and patterns of behaviour. We analysed these further with descriptive and statistical tests using SPSS (version 26). Data are presented as mean (SD). RESULTS: There were 181.44 (75.16) mean daily users accessing the site throughout a 4-month period during 2018/2019. 68.9% of users were female; 44% were 25-34 years; 57.3% used a mobile device. The mean session duration was 73.55 (9.41) seconds, with more time spent per session and a greater number of pages per session observed in users accessing the site from a desktop or tablet as opposed to a mobile phone. 84.3% of mobile users left the site after viewing a single page. Referral source was also associated with device used (p<0.001). Age was not related to user behaviours (p>0.05). DISCUSSION: FOAM is a rapidly developing form of medical education, with large user numbers seen for a site just 2 years old. The site is being used by many beyond its intended readership. Rather than accessing multiple pages from a desktop, users have varied online behaviours, with the majority viewing a single page on a mobile phone, referred by social media or Google. CONCLUSIONS: Google Analytics can powerfully display usage of medical websites but has important limitations if statistical exploration is required. FOAM users are a heterogenous group, and thus content should be designed with this in mind. Further research must be prioritised focussing on the scope, curriculum coverage, accuracy of information and the effectiveness of FOAM as an educational resource.

Hospital Mortality and Trainee Experiences: How General Medical Council Survey Findings Correlate With Summary Hospital-Level Mortality Indicator.

OBJECTIVES: This study aimed to investigate whether components of junior doctor satisfaction are associated with patient mortality within the United Kingdom. METHODS: We conducted a cross-sectional study of publicly available data (the General Medical Council [GMC] National Survey and the Summary Hospital-level Mortality Indicator [SHMI]) pertaining to subjective physician trainee satisfaction and patient mortality within 80 United Kingdom-based healthcare institutions. The direction and strength of correlation between components of the GMC National Survey and relative patient mortality as described by the SHMI were calculated. Additional outcomes included mean GMC survey scores for reported domains and mean SHMI by healthcare institution. RESULTS: SHMI for included healthcare institutions ranged from 0.69 to 1.21 (mean [SD], 1.01 [0.1]). Mean GMC domain scores ranged between 44.61 and 88.62 (mean [SD], 71.16 [10.84]). Statistically significant correlations were observed for clinical supervision, clinical supervision out of hours, rota design, overall satisfaction, and teamwork. After application of Bonferroni correction, statistically significant correlations remained for both clinical supervision and clinical supervision out of hours. CONCLUSIONS: There is a significant association between components of subjective trainee satisfaction and patient mortality within the United Kingdom. Further investigation to examine these relationships, perhaps to target intervention, may prevent avoidable patient harm.

Supervision, scheduling, satisfaction and shared working: how experiences of junior doctors relate to excess mortality within the NHS.

BACKGROUND: We sought to explore associations between trainee doctor perception and excess patient mortality. METHODS: Data from two publicly available databases reflecting mortality and components of trainee satisfaction within 81 NHS healthcare institutions between the years 2012 and 2019 were analysed. Pearson's correlation coefficients were calculated. RESULTS: All domains of trainee perception were correlated with excess mortality. Clinical supervision out of hours (R=-0.44; p<0.0001), teamwork (R=-0.36; p<0.0001) and clinical supervision at any time (R=-0.35; p<0.0001) were most strongly correlated. Most associations remained consistent year on year. CONCLUSION: Trainee doctor perceptions of clinical supervision, rota design and teamwork within the NHS are consistently correlated with excess patient mortality. Further exploration of these associations could identify opportunities for interventions to reduce excess patient mortality. Given the clinical significance of our findings, organisations should consider rapid implementation of evidence-based interventions where they exist.

Between-Sex Differences in Risk Factors for Cardiovascular Disease among Patients with Myocardial Infarction-A Systematic Review.

Between-sex differences in the presentation, risk factors, management, and outcomes of acute myocardial infarction (MI) are well documented. However, as such differences are highly sensitive to cultural and social changes, there is a need to continuously re-evaluate the evidence. The present contemporary systematic review assesses the baseline characteristics of men and women presenting to secondary, tertiary, and quaternary centres with acute myocardial infarction (MI). Over 1.4 million participants from 18 studies, including primary prospective, cross sectional and retrospective observational studies, as well as secondary analysis of registry data are included in the study. The study showed that women were more likely than men to have a previous diagnosis of diabetes, hypertension, cerebrovascular disease, and heart failure. They also had lower odds of presenting with previous ischaemic heart disease and angina, dyslipidaemia, or a smoking history. Further work is necessary to understand the reasons for these differences, and the role that gender-specific risk factors may have in this context. Moreover, how these between-gender differences are implicated in management and outcomes also requires further work.

Ethnic and Regional Differences in the Management of Angina: The Way Forward.

For decades, there has been great interest in ethnic differences in the management of angina and stable cardiovascular disease. Clinical decisionmaking is known to be both consciously and unconsciously influenced by a patient's demographics, and this is due to in part to differences in clinical guidance and opinion. However, the evidence supporting such decision-making is sparse. Nonetheless, there is overwhelming evidence that international, national, regional, institutional, departmental and individual bias disproportionately affect subgroups of the population, resulting in adverse patient outcomes. While without doubt there will be rapid advancements in individualised therapies over the coming years and decades, the most beneficial immediate action clinicians can take is to reduce disparities in both the evidence base and care provision. Doing so will require great collaborative effort.

Racial and ethnic differences in pharmacotherapy to prevent coronary artery disease and thrombotic events.

Awareness of racial/ethnic disparities represents a key challenge for healthcare systems that attempt to provide effective healthcare and to reduce existing inequalities in the use of and adherence to guideline-recommended cardiovascular drugs to improve clinical outcomes for cardiovascular disease (CVD). In this review, we describe important racial/ethnic differences between and within ethnic groups in the prevalence, risk factors, haemostatic factors, anti-inflammatory and endothelial markers, recurrence, and outcomes of CVD. We discuss important differences in the selection, doses, and response [efficacy and adverse drug reactions (ADRs)] in ethnically diverse patients treated with antithrombotics or lipid-lowering drugs. Differences in drug response are mainly related to racial/ethnic differences in the frequency of polymorphisms in genes encoding drug-metabolizing enzymes (DMEs) and drug transporters. These polymorphisms markedly influence the pharmacokinetics, dose requirements, and safety of warfarin, clopidogrel, and statins. This review aims to support a better understanding of the genetic differences between and among populations to identify patients who may experience an ADR or a lack of drug response, thus optimizing therapy and improving outcomes. The greater the understanding of the differences in the genetic variants of DMEs and transporters that determine the differences in the exposure, efficacy, and safety of cardiovascular drugs between races/ethnicities, the greater the probability that personalized medicine will become a reality.

Signatures of recent activation identify a circulating T cell compartment containing tumor-specific antigen receptors with high avidity.

T cell receptor (TCR) avidity is assumed to be a major determinant of the spatiotemporal fate and protective capacity of tumor-specific T cells. However, monitoring polyclonal T cell responses with known TCR avidities in vivo over space and time remains challenging. Here, we investigated the fate and functionality of tumor neoantigen-specific T cells with TCRs of distinct avidities in a well-established, reductionist preclinical tumor model and human patients with melanoma. To this end, we used polyclonal T cell transfers with in-depth characterized TCRs together with flow cytometric phenotyping in mice inoculated with MC38 OVA tumors. Transfer of T cells from retrogenic mice harboring TCRs with high avidity resulted in best tumor protection. Unexpectedly, we found that both high- and low-avidity T cells are similarly abundant within the tumor and adopt concordant phenotypic signs of exhaustion. Outside the tumor, high-avidity TCR T cells were not generally overrepresented but, instead, selectively enriched in T cell populations with intermediate PD-1 protein expression. Single-cell sequencing of neoantigen-specific T cells from two patients with melanoma-combined with transgenic reexpression of identified TCRs by CRISPR-Cas9-mediated orthotopic TCR replacement-revealed high-functionality TCRs to be enriched in T cells with RNA signatures of recent activation. Furthermore, of 130 surface protein candidates, PD-1 surface expression was most consistently enriched in functional TCRs. Together, our findings show that tumor-reactive TCRs with high protective capacity circulating in peripheral blood are characterized by a signature of recent activation.

Medical students' pattern of self-directed learning prior to and during the coronavirus disease 2019 pandemic period and its implications for Free Open Access Meducation within the United Kingdom

PURPOSE: Self-directed learning (SDL) has been increasingly emphasized within medical education. However, little is known about the SDL resources medical students use. This study aimed to identify patterns in medical students' SDL behaviors, their SDL resource choices, factors motivating these choices, and the potential impact of the coronavirus disease 2019 (COVID-19) pandemic on these variables. Methods: An online cross-sectional survey comprising multiple-choice, ranked, and free-text response questions were disseminated to medical students across all 41 UK medical schools between April and July 2020. Independent study hours and sources of study materials prior to and during the COVID-19 pandemic were compared. Motivational factors guiding resource choices and awareness of Free Open Access Meducation were also investigated. Results: The target sample was 75 students per medical school across a total of 41 medical schools within the United Kingdom (3,075 total students), and 1,564 responses were analyzed. University-provided information comprised the most commonly used component of independent study time, but a minority of total independent study time. Independent study time increased as a result of the COVID-19 pandemic (P<0.001). All sub-cohorts except males reported a significant increase in the use of resources such as free websites and question banks (P<0.05) and paid websites (P<0.05) as a result of the pandemic. Accessibility was the most influential factor guiding resource choice (Friedman's µrank=3.97, P<0.001). Conclusion: The use of learning resources independent of university provision is increasing. Educators must ensure equitable access to such materials while supporting students in making informed choices regarding their independent study behaviors.

Antitumor activity without on-target off-tumor toxicity of GD2-chimeric antigen receptor T cells in patients with neuroblastoma.

The reprogramming of a patient's immune system through genetic modification of the T cell compartment with chimeric antigen receptors (CARs) has led to durable remissions in chemotherapy-refractory B cell cancers. Targeting of solid cancers by CAR-T cells is dependent on their infiltration and expansion within the tumor microenvironment, and thus far, fewer clinical responses have been reported. Here, we report a phase 1 study (NCT02761915) in which we treated 12 children with relapsed/refractory neuroblastoma with escalating doses of second-generation GD2-directed CAR-T cells and increasing intensity of preparative lymphodepletion. Overall, no patients had objective clinical response at the evaluation point +28 days after CAR-T cell infusion using standard radiological response criteria. However, of the six patients receiving ≥108/meter2 CAR-T cells after fludarabine/cyclophosphamide conditioning, two experienced grade 2 to 3 cytokine release syndrome, and three demonstrated regression of soft tissue and bone marrow disease. This clinical activity was achieved without on-target off-tumor toxicity. Targeting neuroblastoma with GD2 CAR-T cells appears to be a valid and safe strategy but requires further modification to promote CAR-T cell longevity.

Establishment and phenotyping of neurosphere cultures from primary neuroblastoma samples.

Background: Primary cell culture using serum free media supplemented with growth factors has been used in a number of cancers to propagate primary cells with stem like properties, which form as spherical cellular aggregates. Methods: We systematically evaluated the capacity of freshly disaggregated neuroblastoma tumors to become established as neurospheres in stem cell media using a uniform protocol. 67 primary neuroblastoma samples from patients treated at a single institution were prospectively evaluated for their ability to become established in culture. Samples, either solid tissue or cells from surgical transit fluid both post chemotherapy and chemotherapy naïve, were evaluated from diagnostic needle biopsies or surgical resections. Results: Overall 37 neurosphere cultures were successfully established from 67 samples. In 11 out of 14 cases investigated by flow cytometry, uniform staining for neuroblastoma markers CD56 and GD2 was demonstrated in CD45 negative non-hemopoietic cells, confirming neuroblastoma origin. Conclusion: We present a simple and reproducible approach for producing primary neurospheres from neuroblastoma samples, which provides a reliable resource for future work including genetic analysis, stem cell research and models for therapeutics.

The presence of Y674/Y675 phosphorylated NTRK1 via TP53 repression of PTPN6 expression as a potential prognostic marker in neuroblastoma.

The tumor suppressor TP53 promotes nerve growth factor receptor (NTRK1) -Y674/Y675 phosphorylation (NTRK1-pY674/pY675) via repression of the NTRK1 phosphatase PTPN6 in a ligand-independent manner, resulting in suppression of breast cancer cell proliferation. Moreover, NTRK1-pY674/pY675 together with low levels of PTPN6 and TP53 expression is associated with favorable disease-free survival of breast cancer patients. We determined whether in neuroblastoma this protein expression pattern impacts relapse-free survival (RFS). NTRK1-pY674/pY675, PTPN6, and TP53 expression was assessed in 98 neuroblastoma samples by immunohistochemistry. Association between expression levels and RFS was investigated by multivariate and Kaplan-Meier analysis. Mutant or wild-type TP53 was identified by sequencing tumor DNA. Tumors expressing NTRK1-pY674/pY675 and low or undetectable levels of PTPN6 and TP53 were significantly associated with 5-year RFS (P = .014) when the dataset was stratified by MYCN amplification, segmental chromosomal abnormalities and histology. Similar results were observed with tumors expressing wild-type TP53, NTRK1-pY674/pY675 and low or undetectable levels of PTPN6. Kaplan-Meier analysis demonstrated a significant correlation (P = .004), with a 50% probability of RFS (median survival 4.73 years) when present compared with 19.51% (median survival 11.63 months) when absent. Similar results were seen with non-amplified MYCN or unfavorable/undifferentiating samples and tumors from patients aged 18 months or less. Importantly, NTRK1-pY674/pY675 is an independent predictor of improved RFS. These results strongly suggest that NTRK1-pY674/pY675 together with wild-type TP53 and undetectable or low levels of PTPN6 expression is a potential biomarker of improved RFS of neuroblastoma patients. The predictive value of NTRK1-pY674/pY675 together with wild-type TP53 and low PTPN6 expression could contribute to neuroblastoma patient prognosis.

Language lateralisation measured across linguistic and national boundaries.

The visual half-field technique has been shown to be a reliable and valid neuropsychological measurement of language lateralisation, typically showing higher accuracy and faster correct responses for linguistic stimuli presented in the right visual field (RVF) than left visual field (LVF). The RVF advantage corresponds to the well-known dominance of the left hemisphere (LH) in processing language(s). However, clinical and experimental neuroscientists around the globe use different variations of the visual half-field paradigm, making direct comparisons difficult. The current study used a word/non-word visual half-field paradigm with translingual stimuli. In total, 496 participants from seven European countries were investigated: Belgium (64), England (49), Germany (85), Italy (34), The Netherlands (87), Norway (51), and Switzerland (126), covering six international languages (Dutch, English, French, German, Italian, Norwegian). All language groups revealed a significant RVF/LH advantage in accuracy and reaction times that accounted for up to 26.1% of the total variance in performance. We found some variation in the degree of the RVF/LH advantage across language groups, accounting for a maximum of 3.7% of the total variance in performance. The RVF/LH advantage did not differ between subsamples speaking English, French or German as first or second languages or between monolingual and early/late bi/multilinguals. The findings suggest that the translingual lexical decision task (TLDT) is a simple but reliable measurement of language lateralisation that can be applied clinically and experimentally across linguistic and national boundaries.

Engineered human mesenchymal stem cells for neuroblastoma therapeutics.

Drug-resistant neuroblastoma remains a major challenge in paediatric oncology and novel and less toxic therapeutic approaches are urgently needed to improve survival and reduce the side effects of traditional therapeutic interventions. Mesenchymal stem cells (MSCs) are an attractive candidate for cell and gene therapy since they are recruited by and able to infiltrate tumours. This feature has been exploited by creating genetically modified MSCs that are able to combat cancer by delivering therapeutic molecules. Whether neuroblastomas attract systemically delivered MSCs is still controversial. We investigated whether MSCs engineered to express tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) could: i) cause death of classic and primary neuroblastoma cell lines in vitro; ii) migrate to tumour sites in vivo; and iii) reduce neuroblastoma growth in xenotransplantation experiments. We observed that classic and primary neuroblastoma cell lines expressing death receptors could be killed by TRAIL-loaded MSCs in vitro. When injected in the peritoneum of neuroblastoma-bearing mice, TRAIL-MSCs migrated to tumour sites, but were unable to change the course of cancer development. These results indicated that MSCs have the potential to be used to deliver drugs in neuroblastoma patients, but more effective biopharmaceuticals should be used instead of TRAIL.

Tumor infiltrating lymphocytes expanded from pediatric neuroblastoma display heterogeneity of phenotype and function.

Adoptive transfer of ex vivo expanded tumor infiltrating lymphocytes (TILs) has led to clinical benefit in some patients with melanoma but has not demonstrated convincing efficacy in other solid cancers. Whilst the presence of TILs in many types of cancer is often associated with better clinical prognosis, their function has not been systematically evaluated across cancer types. Responses to immunological checkpoint inhibitors in a wide range of cancers, including those for which adoptive transfer of expanded TILs has not shown clinical benefit, has clearly delineated a number of tumor type associated with tumor-reactive lymphocytes capable of effecting tumor remissions. Neuroblastoma is an aggressive childhood solid cancer in which immunotherapy with GD2-directed antibodies confers a proven survival advantage through incompletely understood mechanisms. We therefore evaluated the feasibility of ex vivo expansion of TILs from freshly resected neuroblastoma tumors and the potential therapeutic utility of TIL expansions. TILs were successfully expanded from both tumor biopsies or resections. Significant numbers of NKT and γδT cells were identified alongside the mixed population of cytotoxic (CD8+) and helper (CD4+) T cells of both effector and central memory phenotypes. Isolated TILs were broadly non-reactive against autologous tumor and neuroblastoma cell lines, so enhancement of neuroblastoma killing was attained by transducing TILs with a second-generation chimeric antigen receptor (CAR) targeting GD2. CAR-TILs demonstrated antigen-specific cytotoxicity against tumor cell lines. This study is the first to show reproducible expansion of TILs from pediatric neuroblastoma, the high proportion of innate-like lymphocytes, and the feasibility to use CAR-TILs therapeutically.

A tailored molecular profiling programme for children with cancer to identify clinically actionable genetic alterations.

BACKGROUND: For children with cancer, the clinical integration of precision medicine to enable predictive biomarker-based therapeutic stratification is urgently needed. METHODS: We have developed a hybrid-capture next-generation sequencing (NGS) panel, specifically designed to detect genetic alterations in paediatric solid tumours, which gives reliable results from as little as 50 ng of DNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue. In this study, we offered an NGS panel, with clinical reporting via a molecular tumour board for children with solid tumours. Furthermore, for a cohort of 12 patients, we used a circulating tumour DNA (ctDNA)-specific panel to sequence ctDNA from matched plasma samples and compared plasma and tumour findings. RESULTS: A total of 255 samples were submitted from 223 patients for the NGS panel. Using FFPE tissue, 82% of all submitted samples passed quality control for clinical reporting. At least one genetic alteration was detected in 70% of sequenced samples. The overall detection rate of clinically actionable alterations, defined by modified OncoKB criteria, for all sequenced samples was 51%. A total of 8 patients were sequenced at different stages of treatment. In 6 of these, there were differences in the genetic alterations detected between time points. Sequencing of matched ctDNA in a cohort of extracranial paediatric solid tumours also identified a high detection rate of somatic alterations in plasma. CONCLUSION: We demonstrate that tailored clinical molecular profiling of both tumour DNA and plasma-derived ctDNA is feasible for children with solid tumours. Furthermore, we show that a targeted NGS panel-based approach can identify actionable genetic alterations in a high proportion of patients.