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There is a growing level of interest in the potential role inflammation has on the initiation and progression of malignancy. Notable examples include Helicobacter pylori -mediated inflammation in gastric cancer and more recently Fusobacterium nucleatum -mediated inflammation in colorectal cancer. Fusobacterium nucleatum is a Gram-negative anaerobic bacterium that was first isolated from the oral cavity and identified as a periodontal pathogen. Biofilms on oral squamous cell carcinomas are enriched with anaerobic periodontal pathogens, including F. nucleatum, which has prompted hypotheses that this bacterium could contribute to oral cancer development. Recent studies have demonstrated that F. nucleatum can promote cancer by several mechanisms; activation of cell proliferation, promotion of cellular invasion, induction of chronic inflammation and immune evasion. This review provides an update on the association between F. nucleatum and oral carcinogenesis, and provides insights into the possible mechanisms underlying it.

Optimisation and validation of a multiplex immunofluorescence (mIF) workflow, from staining to digital image analysis (DIA), ensure assay robustness. Chromogenic immunohistochemistry (IHC) and fluorescent singleplexes are fundamental in this process, particularly when biomarkers are co‐expressed. We describe our experience developing two mIF panels and the various parameters of staining, scanning and DIA to consider when standardising a digital pathology workflow. Multiplex immunofluorescence is a powerful tool for the simultaneous detection of tissue‐based biomarkers, revolutionising traditional immunohistochemistry. The Opal methodology allows up to eight biomarkers to be measured concomitantly without cross‐reactivity, permitting identification of different cell populations within the tumour microenvironment. In this study, we aimed to validate a multiplex immunofluorescence workflow in two complementary multiplex panels and evaluate the tumour immune microenvironment in colorectal cancer (CRC) formalin‐fixed paraffin‐embedded tissue. We stained CRC and tonsil samples using Opal multiplex immunofluorescence on a Leica BOND RX immunostainer. We then acquired images on an Akoya Vectra Polaris and performed multispectral unmixing using inform. Antibody panels were validated on tissue microarray sections containing cores from six normal tissue types, using qupath for image analysis. Comparisons between chromogenic immunohistochemistry and multiplex immunofluorescence on consecutive sections from the same tissue microarray showed significant correlation (rs > 0.9, P‐value < 0.0001), validating both panels. We identified many factors that influenced the quality of the acquired fluorescent images, including biomarker co‐expression, staining order, Opal‐antibody pairing, sample thickness, multispectral unmixing and biomarker detection order during image analysis. Overall, we report the optimisation and validation of a multiplex immunofluorescence process, from staining to image analysis, ensuring assay robustness. Our multiplex immunofluorescence protocols permit the accurate detection of multiple immune markers in various tissue types, using a workflow that enables rapid processing of samples, above and beyond previous workflows.

Background Human Papillomavirus (HPV) is an infectious agent notably associated with viral carcinogenesis of the cervix. Since 2019, the UK and Ireland have used the Gardasil-9 HPV vaccine to prevent new cases of HPV-positive cancers. This systematic review aims to assess whether the current HPV vaccination programme provides substantive protection against developing non-cervical HPV-positive cancers. Methods Relevant studies were identified using the OVID-Medline and EMBASE databases. Screening and data extraction were conducted using the systematic review software Covidence. Risk of bias was assessed using the Hoy et al. tool, and statistical analysis was conducted using R statistical software (v 4.3.1). Results Based on pre-defined search parameters, 4,086 papers were identified for screening. Following the title, abstract, and full-text review, data was extracted from 30 eligible studies. A total of 1,389 patients with HPV-positive cancers, with 24 unique HPV genotypes, were considered for analysis in this review. The most prevalent genotype across all patients was HPV16 (95.9%, 1,332/1,389). Genotypic diversity was notably greater in penile cancers compared to other non-cervical HPV-related cancers considered in the present study, with 21 HPV genotypes reported in this site alone compared to two in vaginal and vulvar cancers ( p  = 1.8E-3). Gardasil-9 was found to offer protection against 37.5% (9/24) of the unique HPV genotypes identified. However, if this vaccine had been available, there would have been sufficient genotype-specific protection to prevent 9 out of 10 HPV-positive cancers (97.8%, 1,359/1,389) retrospectively identified in the current study. It should be noted that significant regional differences in the heterogeneity of reported HPV genotypes (p < 1.0E-05) were illustrated, with London-based cohorts having the most HPV-positive cancers associated with unprotected HPV genotypes (4.7%, 31/658). Conclusion This systematic review confirms HPV16 as the dominant genotype in HPV-positive cancers and highlights the genotypic diversity in non-cervical HPV-positive cancers. Moreover, while HPV vaccination using Gardasil-9 provides limited genotype-specific protection, it could have prevented nearly all HPV-positive cancers reported in eligible studies. Regional differences were minimal, but London had the highest proportion of unprotected genotypes. This suggests that while vaccination is highly effective in preventing HPV-related cancers, gaps in protection remain, particularly for less common genotypes.

Small bowel adenocarcinoma (SBA) is a rare malignancy of the small intestine associated with late stage diagnosis and poor survival outcome. High expression of immune cells and immune checkpoint biomarkers especially programmed cell death ligand-1 (PD-L1) have been shown to significantly impact disease progression. We have analysed the expression of a subset of immune cell and immune checkpoint biomarkers in a cohort of SBA patients and assessed their impact on progression-free survival (PFS) and overall survival (OS). 25 patient samples in the form of formalin fixed, paraffin embedded (FFPE) tissue were obtained in tissue microarray (TMAs) format. Automated immunohistochemistry (IHC) staining was performed using validated antibodies for CD3, CD4, CD8, CD68, PD-L1, ICOS, IDO1 and LAG3. Slides were scanned digitally and assessed in QuPath, an open source image analysis software, for biomarker density and percentage positivity. Survival analyses were carried out using the Kaplan Meier method. Varying expressions of biomarkers were recorded. High expressions of CD3, CD4 and IDO1 were significant for PFS (p = 0.043, 0.020 and 0.018 respectively). High expression of ICOS was significant for both PFS (p = 0.040) and OS (p = 0.041), while high PD-L1 expression in tumour cells was significant for OS (p = 0.033). High correlation was observed between PD-L1 and IDO1 expressions (Pearson correlation co-efficient = 1) and subsequently high IDO1 expression in tumour cells was found to be significant for PFS (p = 0.006) and OS (p = 0.034). High levels of immune cells and immune checkpoint proteins have a significant impact on patient survival in SBA. These data could provide an insight into the immunotherapeutic management of patients with SBA.

Purpose of the Review The purpose of the current paper was to review and summarize the literature on ADHD and maltreatment over the past 10 years. Recent Findings The majority of research on ADHD and exposure to maltreatment focuses on the high rates of comorbidity, including international studies from Asia, South America, North America, and Europe. Longitudinal studies showed that early exposure to maltreatment is a risk factor for ADHD symptoms later in development; however, this finding was not consistent. There were some preliminary studies on the neurological and genetic mechanisms underlying the link between ADHD and exposure to maltreatment. Finally, ADHD and exposure to maltreatment were found to have an additive effect on clinically salient outcomes (e.g., aggression, suicide attempts). Summary Results from the review have direct clinical and future implications, including the need to understand the effect of comorbid ADHD and exposure to maltreatment in treatment studies.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal human malignancies. Tissue microarrays (TMA) are an established method of high throughput biomarker interrogation in tissues but may not capture histological features of cancer with potential biological relevance. Topographic TMAs (T-TMAs) representing pathophysiological hallmarks of cancer were constructed from representative, retrospective PDAC diagnostic material, including 72 individual core tissue samples. The T-TMA was interrogated with tissue hybridization-based experiments to confirm the accuracy of the topographic sampling, expression of pro-tumourigenic and immune mediators of cancer, totalling more than 750 individual biomarker analyses. A custom designed Next Generation Sequencing (NGS) panel and a spatial distribution-specific transcriptomic evaluation were also employed. The morphological choice of the pathophysiological hallmarks of cancer was confirmed by protein-specific expression. Quantitative analysis identified topography-specific patterns of expression in the IDO/TGF-β axis; with a heterogeneous relationship of inflammation and desmoplasia across hallmark areas and a general but variable protein and gene expression of c-MET. NGS results highlighted underlying genetic heterogeneity within samples, which may have a confounding influence on the expression of a particular biomarker. T-TMAs, integrated with quantitative biomarker digital scoring, are useful tools to identify hallmark specific expression of biomarkers in pancreatic cancer.

Background Limited studies examine the immune landscape in Esophageal Adenocarcinoma (EAC). We aim to identify novel associations, which may inform immunotherapy treatment stratification. Methods Three hundred twenty-nine EAC cases were available in Tissue Microarrays (TMA) format. A discovery cohort of 166 EAC cases were stained immunohistochemically for range of adaptive immune (CD3, CD4, CD8 and CD45RO) and immune checkpoint biomarkers (ICOS, IDO-1, PD-L1, PD-1). A validation cohort of 163 EAC cases was also accessed. A digital pathology analysis approach was used to quantify biomarker density. Results CD3, CD4, CD8, CD45RO, ICOS and PD-1 were individually predictive of better overall survival (OS) (Log rank p  = < 0.001; p  = 0.014; p  = 0.001; p  = < 0.001; p  = 0.008 and p  = 0.026 respectively). Correlation and multivariate analysis identified high CD45RO/ICOS patients with significantly improved OS which was independently prognostic (HR = 0.445, (0.223–0.886), p  = 0.021). Assessment of CD45RO and ICOS high cases in the validation cohort revealed an associated with improved OS (HR = 0.601 (0.363–0.996), p  = 0.048). Multiplex IHC identified cellular co-expression of high CD45RO/ICOS. High CD45RO/ICOS patients have significantly improved OS. Conclusions Multiplexing identifies true cellular co-expression. These data demonstrate that co-expression of immune biomarkers are associated with better outcome in EAC and may provide evidence for immunotherapy treatment stratification.

Background The cohesin complex plays a major role in folding the human genome into 3D structural domains. Mutations in members of the cohesin complex are known early drivers of myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML), with STAG2 the most frequently mutated complex member. Methods Here we use functional genomics (RNA-seq, ChIP-seq and HiChIP) to investigate the impact of chronic STAG2 loss on three-dimensional genome structure and transcriptional programming in a clinically relevant model of chronic STAG2 loss. Results The chronic loss of STAG2 led to loss of smaller loop domains and the maintenance/formation of large domains that, in turn, led to altered genome compartmentalisation. These changes in genome structure resulted in altered gene expression, including deregulation of the HOXA locus and the MAPK signalling pathway, resulting in increased sensitivity to MEK inhibition. Conclusions The altered genomic architecture driven by the chronic loss of STAG2 results in altered gene expression that may contribute to leukaemogenesis and may be therapeutically targeted.

IntroductionAdolescence represents a critical developmental period, with changes in emotional regulation capacities influencing physical and mental health. With less than 6% of Canadian youth currently meeting the 24-hour movement guidelines for physical activity, sleep and sedentary behaviour, there is an urgent need to understand the potential association between movement behaviours, physical literacy, emotional regulation and mental health during adolescence. Additionally, there is a need to better understand these associations among equity-deserving groups. We developed the Adolescents’ Daily Lives (ADL) project to identify how, when, under what contexts and to whom to promote healthy engagement in movement behaviours to optimise youth mental health.Methods and analysisFor the ADL project, we will employ a 14-day intensive longitudinal design to investigate the associations between physical literacy, movement behaviours, emotion regulation and mental health among a diverse sample of 120 adolescents (ages 13–17 years) living in the Greater Victoria Area, British Columbia, Canada. A comprehensive baseline survey and movement competence test, assessing physical and mental well-being, 24-hour movement behaviours (ie, physical activity, sleep and sedentary behaviours) and physical literacy, will be accompanied by daily diary surveys and accelerometer-based movement tracking (ie, Fitbit Inspire 3) to assess daily fluctuations in movement behaviour, emotional regulation and mood. Multivariate analyses, including multilevel modelling, multilevel structural equation modelling and Bayesian hierarchical continuous-time SEM, will be used to model the repeated measures data and understand the simultaneous variations in daily movement behaviours, emotion regulation and mental health.Ethics and disseminationThe ADL project received ethical approval from the University of Victoria Behavioural Research Ethics Board (protocol #22-0262). Study participation is voluntary, and data collection will be anonymised to protect participant privacy and confidentiality. Research findings will be shared through academic publications and conference proceedings. Through knowledge mobilisation resources, cocreated with the youth community advisory board, relevant findings will be shared directly with the wider community of adolescents.

Oral epithelial dysplasia (OED) is a premalignant histopathological diagnosis given to lesions of the oral cavity. Its grading suffers from significant inter-/intra-observer variability, and does not reliably predict malignancy progression, potentially leading to suboptimal treatment decisions. To address this, we developed an artificial intelligence (AI) algorithm, that assigns an Oral Malignant Transformation (OMT) risk score based on the Haematoxylin and Eosin (H&E) stained whole slide images (WSIs). Our AI pipeline leverages an in-house segmentation model to detect and segment both nuclei and epithelium. Subsequently, a shallow neural network utilises interpretable morphological and spatial features, emulating histological markers, to predict progression. We conducted internal cross-validation on our development cohort (Sheffield; n  = 193 cases) and independent validation on two external cohorts (Birmingham and Belfast; n  = 89 cases). On external validation, the proposed OMTscore achieved an AUROC = 0.75 (Recall = 0.92) in predicting OED progression, outperforming other grading systems (Binary: AUROC = 0.72, Recall = 0.85). Survival analyses showed the prognostic value of our OMTscore (C-index = 0.60, p  = 0.02), compared to WHO (C-index = 0.64, p  = 0.003) and binary grades (C-index = 0.65, p  < 0.001). Nuclear analyses elucidated the presence of peri-epithelial and intra-epithelial lymphocytes in highly predictive patches of transforming cases ( p  < 0.001). This is the first study to propose a completely automated, explainable, and externally validated algorithm for predicting OED transformation. Our algorithm shows comparable-to-human-level performance, offering a promising solution to the challenges of grading OED in routine clinical practice.