TH9 cells that express the transcription factor PU.1 drive T cell-mediated colitis via IL-9 receptor signaling in intestinal epithelial cells.

The molecular checkpoints that drive inflammatory bowel diseases are incompletely understood. Here we found more T cells expressing the transcription factor PU.1 and interleukin 9 (IL-9) in patients with ulcerative colitis. In an animal model, citrine reporter mice had more IL-9-expressing mucosal T cells in experimental oxazolone-induced colitis. IL-9 deficiency suppressed acute and chronic colitis. Mice with PU.1 deficiency in T cells were protected from colitis, whereas treatment with antibody to IL-9 suppressed colitis. Functionally, IL-9 impaired intestinal barrier function and prevented mucosal wound healing in vivo. Thus, our findings suggest that the TH9 subset of helper T cells serves an important role in driving ulcerative colitis by regulating intestinal epithelial cells and that TH9 cells represent a likely target for the treatment of chronic intestinal inflammation.

Image-based multiplex immune profiling of cancer tissues: translational implications. A report of the International Immuno-oncology Biomarker Working Group on Breast Cancer.

Recent advances in the field of immuno-oncology have brought transformative changes in the management of cancer patients. The immune profile of tumours has been found to have key value in predicting disease prognosis and treatment response in various cancers. Multiplex immunohistochemistry and immunofluorescence have emerged as potent tools for the simultaneous detection of multiple protein biomarkers in a single tissue section, thereby expanding opportunities for molecular and immune profiling while preserving tissue samples. By establishing the phenotype of individual tumour cells when distributed within a mixed cell population, the identification of clinically relevant biomarkers with high-throughput multiplex immunophenotyping of tumour samples has great potential to guide appropriate treatment choices. Moreover, the emergence of novel multi-marker imaging approaches can now provide unprecedented insights into the tumour microenvironment, including the potential interplay between various cell types. However, there are significant challenges to widespread integration of these technologies in daily research and clinical practice. This review addresses the challenges and potential solutions within a structured framework of action from a regulatory and clinical trial perspective. New developments within the field of immunophenotyping using multiplexed tissue imaging platforms and associated digital pathology are also described, with a specific focus on translational implications across different subtypes of cancer. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

The genetic regulation of the gastric transcriptome is associated with metabolic and obesity-related traits and diseases.

Tissue-specific gene expression and gene regulation lead to a better understanding of tissue-specific physiology and pathophysiology. We analyzed the transcriptome and genetic regulatory profiles of two distinct gastric sites, corpus and antrum, to identify tissue-specific gene expression and its regulation. Gastric corpus and antrum mucosa biopsies were collected during routine gastroscopies from up to 431 healthy individuals. We obtained genotype and transcriptome data and performed transcriptome profiling and expression quantitative trait locus (eQTL) studies. We further used data from genome-wide association studies (GWAS) of various diseases and traits to partition their heritability and to perform transcriptome-wide association studies (TWAS). The transcriptome data from corpus and antral mucosa highlights the heterogeneity of gene expression in the stomach. We identified enriched pathways revealing distinct and common physiological processes in gastric corpus and antrum. Furthermore, we found an enrichment of the single nucleotide polymorphism (SNP)-based heritability of metabolic, obesity-related, and cardiovascular traits and diseases by considering corpus- and antrum-specifically expressed genes. Particularly, we could prioritize gastric-specific candidate genes for multiple metabolic traits, like NQO1 which is involved in glucose metabolism, MUC1 which contributes to purine and protein metabolism or RAB27B being a regulator of weight and body composition. Our findings show that gastric corpus and antrum vary in their transcriptome and genetic regulatory profiles indicating physiological differences which are mostly related to digestion and epithelial protection. Moreover, our findings demonstrate that the genetic regulation of the gastric transcriptome is linked to biological mechanisms associated with metabolic, obesity-related, and cardiovascular traits and diseases. NEW & NOTEWORTHY We analyzed the transcriptomes and genetic regulatory profiles of gastric corpus and for the first time also of antrum mucosa in 431 healthy individuals. Through tissue-specific gene expression and eQTL analyses, we uncovered unique and common physiological processes across both primary gastric sites. Notably, our findings reveal that stomach-specific eQTLs are enriched in loci associated with metabolic traits and diseases, highlighting the pivotal role of gene expression regulation in gastric physiology and potential pathophysiology.

CagA-specific Gastric CD8+ Tissue-Resident T Cells Control Helicobacter pylori During the Early Infection Phase.

BACKGROUND & AIMS: Infection with Helicobacter pylori strongly affects global health by causing chronic gastritis, ulcer disease, and gastric cancer. Although extensive research into the strong immune response against this persistently colonizing bacterium exists, the specific role of CD8+ T cells remains elusive. METHODS: We comprehensively characterize gastric H pylori-specific CD8+ T-cell responses in mice and humans by flow cytometry, RNA-sequencing, immunohistochemistry, and ChipCytometry, applying functional analyses including T-cell depletion, H pylori eradication, and ex vivo restimulation. RESULTS: We define CD8+ T-cell populations bearing a tissue-resident memory (TRM) phenotype, which infiltrate the gastric mucosa shortly after infection and mediate pathogen control by executing antigen-specific effector properties. These induced CD8+ tissue-resident memory T cells (TRM cells) show a skewed T-cell receptor beta chain usage and are mostly specific for cytotoxin-associated gene A, the distinctive oncoprotein injected by H pylori into host cells. As the infection progresses, we observe a loss of the TRM phenotype and replacement of CD8+ by CD4+ T cells, indicating a shift in the immune response during the chronic infection phase. CONCLUSIONS: Our results point toward a hitherto unknown role of CD8+ T-cell response in this bacterial infection, which may have important clinical implications for treatment and vaccination strategies against H pylori.

Artificial Intelligence Enabled Histological Prediction of Remission or Activity and Clinical Outcomes in Ulcerative Colitis.

BACKGROUND & AIMS: Microscopic inflammation has significant prognostic value in ulcerative colitis (UC); however, its assessment is complex with high interobserver variability. We aimed to develop and validate an artificial intelligence (AI) computer-aided diagnosis system to evaluate UC biopsies and predict prognosis. METHODS: A total of 535 digitalized biopsies (273 patients) were graded according to the PICaSSO Histologic Remission Index (PHRI), Robarts, and Nancy Histological Index. A convolutional neural network classifier was trained to distinguish remission from activity on a subset of 118 biopsies, calibrated on 42 and tested on 375. The model was additionally tested to predict the corresponding endoscopic assessment and occurrence of flares at 12 months. The system output was compared with human assessment. Diagnostic performance was reported as sensitivity, specificity, prognostic prediction through Kaplan-Meier, and hazard ratios of flares between active and remission groups. We externally validated the model in 154 biopsies (58 patients) with similar characteristics but more histologically active patients. RESULTS: The system distinguished histological activity/remission with sensitivity and specificity of 89% and 85% (PHRI), 94% and 76% (Robarts Histological Index), and 89% and 79% (Nancy Histological Index). The model predicted the corresponding endoscopic remission/activity with 79% and 82% accuracy for UC endoscopic index of severity and Paddington International virtual ChromoendoScopy ScOre, respectively. The hazard ratio for disease flare-up between histological activity/remission groups according to pathologist-assessed PHRI was 3.56, and 4.64 for AI-assessed PHRI. Both histology and outcome prediction were confirmed in the external validation cohort. CONCLUSION: We developed and validated an AI model that distinguishes histologic remission/activity in biopsies of UC and predicts flare-ups. This can expedite, standardize, and enhance histologic assessment in practice and trials.

International consensus on the management of metastatic gastric cancer: step by step in the foggy landscape : Bertinoro Workshop, November 2022.

BACKGROUND: Many gastric cancer patients in Western countries are diagnosed as metastatic with a median overall survival of less than twelve months using standard chemotherapy. Innovative treatments, like targeted therapy or immunotherapy, have recently proved to ameliorate prognosis, but a general agreement on managing oligometastatic disease has yet to be achieved. An international multi-disciplinary workshop was held in Bertinoro, Italy, in November 2022 to verify whether achieving a consensus on at least some topics was possible. METHODS: A two-round Delphi process was carried out, where participants were asked to answer 32 multiple-choice questions about CT, laparoscopic staging and biomarkers, systemic treatment for different localization, role and indication of palliative care. Consensus was established with at least a 67% agreement. RESULTS: The assembly agreed to define oligometastases as a "dynamic" disease which either regresses or remains stable in response to systemic treatment. In addition, the definition of oligometastases was restricted to the following sites: para-aortic nodal stations, liver, lung, and peritoneum, excluding bones. In detail, the following conditions should be considered as oligometastases: involvement of para-aortic stations, in particular 16a2 or 16b1; up to three technically resectable liver metastases; three unilateral or two bilateral lung metastases; peritoneal carcinomatosis with PCI ≤ 6. No consensus was achieved on how to classify positive cytology, which was considered as oligometastatic by 55% of participants only if converted to negative after chemotherapy. CONCLUSION: As assessed at the time of diagnosis, surgical treatment of oligometastases should aim at R0 curativity on the entire disease volume, including both the primary tumor and its metastases. Conversion surgery was defined as surgery on the residual volume of disease, which was initially not resectable for technical and/or oncological reasons but nevertheless responded to first-line treatment.

Dysplastic crypt-rings in tandem: A novel histologic parameter in tubular adenomas.

Descriptions of the various dysplastic crypt phenotypes occurring in TA have remained unattended in the literature. Recently, new crypt-phenotypes, characterized by crypt rings in tandem (CRT), and by dysplastic crypt rings in tandem (DCRT) were described in IBD, and in in IBD-associated dysplasia, respectively. Here, we report the occurrence of DCRT in 40.4 % (n = 59) out of 146 consecutive tubular adenomas of the colorectum (TA). The number of DCRT varied: 10 TA had two DCRT, seven TA had three DCRT, two TA, four DCRT and the remaining two TA had ≥ five DCRT. The frequency of DCRT was influenced by TA-size; larger TA (≥ 5 mm) had significantly more DCRT than smaller TA (<5 mm). Conversely, the frequency of TA with DCRT was not influenced by age, gender, or localization. Since only 1 or 2 sections were available per TA, the number of DCRT in the entire TA should be higher than those shown in Results. Historical controls in human and rodent normal colorectum showed no CRT. Moreover, DCRT were not found in 781 historical non-polypoid colorectal adenomas. The present finding might encourage searching for DCRT, the final goal being to achieve a more elaborated microscopic narrative of TA, the most prevalent of all colorectal adenomas.

International consensus on the management of large (≥20 mm) colorectal laterally spreading tumors: World Endoscopy Organization Delphi study.

OBJECTIVES: There have been significant advances in the management of large (≥20 mm) laterally spreading tumors (LSTs) or nonpedunculated colorectal polyps; however, there is a lack of clear consensus on the management of these lesions with significant geographic variability especially between Eastern and Western paradigms. We aimed to provide an international consensus to better guide management and attempt to homogenize practices. METHODS: Two experts in interventional endoscopy spearheaded an evidence-based Delphi study on behalf of the World Endoscopy Organization Colorectal Cancer Screening Committee. A steering committee comprising six members devised 51 statements, and 43 experts from 18 countries on six continents participated in a three-round voting process. The Grading of Recommendations, Assessment, Development and Evaluations tool was used to assess evidence quality and recommendation strength. Consensus was defined as ≥80% agreement (strongly agree or agree) on a 5-point Likert scale. RESULTS: Forty-two statements reached consensus after three rounds of voting. Recommendations included: three statements on training and competency; 10 statements on preresection evaluation, including optical diagnosis, classification, and staging of LSTs; 14 statements on endoscopic resection indications and technique, including statements on en bloc and piecemeal resection decision-making; seven statements on postresection evaluation; and eight statements on postresection care. CONCLUSIONS: An international expert consensus based on the current available evidence has been developed to guide the evaluation, resection, and follow-up of LSTs. This may provide guiding principles for the global management of these lesions and standardize current practices.

Different levels of healing in inflammatory bowel diseases: mucosal, histological, transmural, barrier and complete healing.

Mucosal healing on endoscopy has emerged as a key prognostic parameter in the management of patients with IBD (Crohn's disease, ulcerative colitis/UC) and can predict sustained clinical remission and resection-free survival. The structural basis for this type of mucosal healing is a progressive resolution of intestinal inflammation with associated healing of ulcers and improved epithelial barrier function. However, in some cases with mucosal healing on endoscopy, evidence of histological activity in mucosal biopsies has been observed. Subsequently, in UC, a second, deeper type of mucosal healing, denoted histological healing, was defined which requires the absence of active inflammation in mucosal biopsies. Both levels of mucosal healing should be considered as initial events in the resolution of gut inflammation in IBD rather than as indicators of complete transmural healing. In this review, the effects of anti-inflammatory, biological or immunosuppressive agents as well as small molecules on mucosal healing in clinical studies are highlighted. In addition, we focus on the implications of mucosal healing for clinical management of patients with IBD. Moreover, emerging techniques for the analysis of mucosal healing as well as potentially deeper levels of mucosal healing such as transmural healing and functional barrier healing of the mucosa are discussed. Although none of these new levels of healing indicate a definitive cure of the diseases, they make an important contribution to the assessment of patients' prognosis. The ultimate level of healing in IBD would be a resolution of all aspects of intestinal and extraintestinal inflammation (complete healing).

Helicobacter pylori promotes colorectal carcinogenesis by deregulating intestinal immunity and inducing a mucus-degrading microbiota signature.

OBJECTIVE: Helicobacter pylori infection is the most prevalent bacterial infection worldwide. Besides being the most important risk factor for gastric cancer development, epidemiological data show that infected individuals harbour a nearly twofold increased risk to develop colorectal cancer (CRC). However, a direct causal and functional connection between H. pylori infection and colon cancer is lacking. DESIGN: We infected two Apc-mutant mouse models and C57BL/6 mice with H. pylori and conducted a comprehensive analysis of H. pylori-induced changes in intestinal immune responses and epithelial signatures via flow cytometry, chip cytometry, immunohistochemistry and single cell RNA sequencing. Microbial signatures were characterised and evaluated in germ-free mice and via stool transfer experiments. RESULTS: H. pylori infection accelerated tumour development in Apc-mutant mice. We identified a unique H. pylori-driven immune alteration signature characterised by a reduction in regulatory T cells and pro-inflammatory T cells. Furthermore, in the intestinal and colonic epithelium, H. pylori induced pro-carcinogenic STAT3 signalling and a loss of goblet cells, changes that have been shown to contribute-in combination with pro-inflammatory and mucus degrading microbial signatures-to tumour development. Similar immune and epithelial alterations were found in human colon biopsies from H. pylori-infected patients. Housing of Apc-mutant mice under germ-free conditions ameliorated, and early antibiotic eradication of H. pylori infection normalised the tumour incidence to the level of uninfected controls. CONCLUSIONS: Our studies provide evidence that H. pylori infection is a strong causal promoter of colorectal carcinogenesis. Therefore, implementation of H. pylori status into preventive measures of CRC should be considered.

Monitoring Patients With Eosinophilic Esophagitis in Routine Clinical Practice - International Expert Recommendations.

BACKGROUND & AIMS: There are no studies or recommendations on optimal monitoring strategies for patients with eosinophilic esophagitis (EoE). Our objective was to develop guidance on how to monitor patients with EoE in routine clinical practice, on the basis of available clinical evidence and expert opinion. METHODS: A multidisciplinary, international group of EoE experts identified the following important 3 questions during several consensus meetings: why, by what means, and when to monitor patients with EoE. A steering committee was named, and 3 teams were formed to review literature and to formulate statements for each topic. In a Delphi survey, a level of agreement of ≥75% was defined as threshold value for acceptance. In a final conference, results were presented, critical points and comments on the statements were discussed, and statements were rephrased/rewritten if necessary. RESULTS: Eighteen EoE experts (14 adult and pediatric gastroenterologists, 2 pathologists and 2 allergists) with a median of 21.7 years in clinical practice, mostly academic or university-based, completed the Delphi survey, which included 11 statements and a proposed algorithm for monitoring patients with EoE. Each statement attained ≥75% agreement. Participants discussed and debated mostly about the statement concerning surveillance intervals for EoE patients with stable disease. CONCLUSIONS: It was concluded that effective maintenance treatment probably reduces the development of EoE complications, and regular, structured, and, under certain conditions, individualized clinical follow-up is recommended to assess disease activity while opening a window to monitoring side effects, adjusting therapy, and encouraging adherence to treatment. Follow-up should comprise symptom assessment and periodic or repeated endoscopy with histological assessment in specific EoE settings.

Fully Automated Tumor Bud Assessment in Hematoxylin and Eosin-Stained Whole Slide Images of Colorectal Cancer.

Tumor budding (TB), the presence of single cells or small clusters of up to 4 tumor cells at the invasive front of colorectal cancer (CRC), is a proven risk factor for adverse outcomes. International definitions are necessary to reduce interobserver variability. According to the current international guidelines, hotspots at the invasive front should be counted in hematoxylin and eosin (H&E)-stained slides. This is time-consuming and prone to interobserver variability; therefore, there is a need for computer-aided diagnosis solutions. In this study, we report an artificial intelligence-based method for detecting TB in H&E-stained whole slide images. We propose a fully automated pipeline to identify the tumor border, detect tumor buds, characterize them based on the number of tumor cells, and produce a TB density map to identify the TB hotspot. The method outputs the TB count in the hotspot as a computational biomarker. We show that the proposed automated TB detection workflow performs on par with a panel of 5 pathologists at detecting tumor buds and that the hotspot-based TB count is an independent prognosticator in both the univariate and the multivariate analysis, validated on a cohort of n = 981 patients with CRC. Computer-aided detection of tumor buds based on deep learning can perform on par with expert pathologists for the detection and quantification of tumor buds in H&E-stained CRC histopathology slides, strongly facilitating the introduction of budding as an independent prognosticator in clinical routine and clinical trials.

The frequency of dysplastic branching crypts in colorectal polypoid tubular adenomas.

Dysplastic crypt branching (DCB) was recently found in ulcerative colitis-associated dysplasia. The aim was to assess the frequency and the branching phenotype of DCB in polypoid colorectal tubular adenomas (TA). A total of 3956 DCB were found in the 139 TA: 98% were in asymmetric branching (DCAB) and the remaining 2% in symmetric branching (DCSB). A linear correlation was found between DCB frequency and the increasing digital size in TA (p < .05). Using a digital ruler, adenomas were divided into small TA (<5 mm) and larger TA (≥5 mm). The difference between the frequency of DCB in small TA (n = 75) vs. larger TA (n = 64), was significant (p < .05). DCB frequency was not influenced by age, gender or TA localization. In the normal colorectal mucosa (≈2 m2 ), only occasional CSB is found and no CAB. And yet, multiple DCB (mean 16.7 DCB), mostly DCAB, was found in small TA, occupying <5 mm of the mucosal area. In larger TA, as many as 42.1 DCB (mean), mostly DCAB, occurred in merely 7.8 mm (mean) of the colon mucosa. Thus it is suggested that DCB is a standard histologic element of TA. The natural expansion of the adenomatous tissue in larger TA appears to be follow on from newly produced, mostly DCAB, by DCSB and by the accumulation of their dysplastic offspring's progenies. The findings strongly suggest that DCB is a central microstructure in the histological events unfolding in polypoid colorectal TA.

Endoscopic submucosal dissection techniques and technology: European Society of Gastrointestinal Endoscopy (ESGE) Technical Review.

ESGE suggests conventional endoscopic submucosal dissection (ESD; marking and mucosal incision followed by circumferential incision and stepwise submucosal dissection) for most esophageal and gastric lesions. ESGE suggests tunneling ESD for esophageal lesions involving more than two-thirds of the esophageal circumference. ESGE recommends the pocket-creation method for colorectal ESD, at least if traction devices are not used. The use of dedicated ESD knives with size adequate to the location/thickness of the gastrointestinal wall is recommended. It is suggested that isotonic saline or viscous solutions can be used for submucosal injection. ESGE recommends traction methods in esophageal and colorectal ESD and in selected gastric lesions. After gastric ESD, coagulation of visible vessels is recommended, and post-procedural high dose proton pump inhibitor (PPI) (or vonoprazan). ESGE recommends against routine closure of the ESD defect, except in duodenal ESD. ESGE recommends corticosteroids after resection of  > 50 % of the esophageal circumference. The use of carbon dioxide when performing ESD is recommended. ESGE recommends against the performance of second-look endoscopy after ESD. ESGE recommends endoscopy/colonoscopy in the case of significant bleeding (hemodynamic instability, drop in hemoglobin > 2 g/dL, severe ongoing bleeding) to perform endoscopic hemostasis with thermal methods or clipping; hemostatic powders represent rescue therapies. ESGE recommends closure of immediate perforations with clips (through-the-scope or cap-mounted, depending on the size and shape of the perforation), as soon as possible but ideally after securing a good plane for further dissection.

A20 undermines alternative NF-κB activity and expression of anti-apoptotic genes in Helicobacter pylori infection.

A hallmark of infection by the pathogen Helicobacter pylori, which colonizes the human gastric epithelium, is the simultaneous activation of the classical and alternative nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways, underlying inflammation and cell survival. Here, we report that the classical NF-κB target gene product A20 contributes to the negative regulation of alternative NF-κB signaling in gastric epithelial cells infected by H. pylori. Mechanistically, the de novo synthesized A20 protein interacts with tumor necrosis factor receptor-associated factor-interacting protein with forkhead-associated domain (TIFA) and thereby interferes with the association of TIFA with the NIK regulatory complex. We also show that alternative NF-κB activity contributes to the up-regulation of anti-apoptotic genes, such as baculoviral IAP repeat containing 2 (BIRC2), BIRC3 and B-cell lymphoma 2-related protein A1 (BCL2A1) in gastric epithelial cells. Furthermore, the observed over-expression of RelB in human gastric biopsies with type B gastritis and RelB-dependent suppression of apoptotic cell death emphasize an important role of the alternative NF-κB pathway in H. pylori infection.

Detection of Fusobacterium nucleatum in Patients with Colitis-Associated Colorectal Cancer.

Fusobacterium nucleatum is supposed to play a critical role in the development of colorectal cancer. The species has also been associated with ulcerative colitis (UC) that can progress into colorectal cancer, however, the involvement of bacteria in this process remains unclear. We analysed 177 colon biopsies obtained from patients during screening, including 20 healthy controls, 56 UC cases and 69 cases at different stages of progression to colitis-associated cancer (CAC); 32 samples of sporadic colorectal carcinoma (sCRC) were also included. The presence of F. nucleatum was detected by quantitative real-time PCR (qPCR). Our data show an association between the presence of the bacteria and the progression of carcinogenesis in UC patients. In 39.5% of CAC samples F. nucleatum was detected, compared to only 1.8% in UC cases. The bacteria were detected in 6.3% of samples with initial neoplastic transformation, so-called low-grade dysplasia (LGD), whereas high-grade dysplasia (HGD) resulted in 33.3% of samples positive for F. nucleatum. The fraction of F. nucleatum-positive samples from sCRC cases was 56.3%, which was not significantly different to the CAC group. We conclude that F. nucleatum is associated with the occurrence and progression of colon carcinogenesis, rather than with UC itself.

Paravertebral fibrous pseudotumor: Four cases of a distinctive tumefactive lesion overlapping with eosinophilic angiocentric fibrosis and tumoral erythema elevatum diutinum.

Eosinophilic angiocentric fibrosis (EAF) is a rare tumefactive fibroinflammatory disease with predilection for the upper respiratory tract, characterized by concentric (onionskin) fibrosis around small arterioles with variable intervening storiform fibrosis admixed with chronic inflammatory infiltrates rich in eosinophils. Erythema elevatum diutinum (EED), another autoimmunological disorder that mainly affects acral sites and extensor surfaces, is characterized by neutrophilic leukocytoclastic vasculitis. Rarely, older EED lesions may present as tumefactive nodular (pseudotumoral) fibrous masses closely mimicking EAF. We herein describe four patients (all males) aged 66-70 years who presented with large (median, 7 cm) tumor-like fibrous lesions in the paravertebral region not associated with a known clinical autoimmune disease. All cases were resected surgically with the suspicion of a neoplasm. They displayed a strikingly similar histological appearance with combined features of EAF and nodular fibrous EED. None had evidence of obliterative phlebitis or increased IgG4: IgG ratio. The etiology of this distinctive lesion and its predilection for the paravertebral area of males remains obscure. A distinctive tumefactive localized reaction to trauma caused by degenerative disease of adjacent vertebrae might be a possible explanation.

Possible tumour cell reimplantation during curative endoscopic therapy of superficial Barrett's carcinoma.

BACKGROUND AND AIMS: Endoscopic resection has been established as curative therapy for superficial cancer arising from Barrett's oesophagus (BE); recurrences are very rare. Based on a case series with unusual and massive early recurrences, we analyse the issue of tumour cell reimplantation. METHODS: This hypothesis was developed on the basis of two out of seven patients treated by circumferential (n=6) or nearly circumferential (n=1) en bloc and R0 endoscopic resection of T1 neoplastic BE. Subsequently, a prospective histocytological analysis of endoscope channels and accessories was performed in 2 phases (cytohistological analysis; test for cell viability) in 22 different oesophageal carcinoma patients undergoing endoscopy. Finally, cultures from two oesophageal adenocarcinoma cell lines were incubated with different triamcinolone concentrations (0.625-10 mg/mL); cell growth was determined on a Multiwell plate reader. RESULTS: Cancer regrowth in the two suspicious cases (male, 78/71 years) occurred 7 and 1 months, respectively, after curative tumour resection. Subsequent surgery showed advanced tumours (T2) with lymph node metastases; one patient died. On cytohistological examinations of channels and accessories, suspicious/neoplastic cells were found in 4/10 superficial and in all 5 advanced cancers. Further analyses in seven further advanced adenocarcinoma cases showed viable cells in two channel washing specimens. Finally, cell culture experiments demonstrated enhanced tumour cell growth by triamcinolone after 24 hours compared with controls. CONCLUSIONS: Tumour cell reimplanation from contaminated endoscopes and accessories is a possible cause of local recurrence after curative endoscopic therapy for superficial Barrett carcinoma; also, corticosteroid injection could have promoted tumour regrowth in these cases.

Neutrophils prevent rectal bleeding in ulcerative colitis by peptidyl-arginine deiminase-4-dependent immunothrombosis.

OBJECTIVE: Bleeding ulcers and erosions are hallmarks of active ulcerative colitis (UC). However, the mechanisms controlling bleeding and mucosal haemostasis remain elusive. DESIGN: We used high-resolution endoscopy and colon tissue samples of active UC (n = 36) as well as experimental models of physical and chemical mucosal damage in mice deficient for peptidyl-arginine deiminase-4 (PAD4), gnotobiotic mice and controls. We employed endoscopy, histochemistry, live-cell microscopy and flow cytometry to study eroded mucosal surfaces during mucosal haemostasis. RESULTS: Erosions and ulcerations in UC were covered by fresh blood, haematin or fibrin visible by endoscopy. Fibrin layers rather than fresh blood or haematin on erosions were inversely correlated with rectal bleeding in UC. Fibrin layers contained ample amounts of neutrophils coaggregated with neutrophil extracellular traps (NETs) with detectable activity of PAD. Transcriptome analyses showed significantly elevated PAD4 expression in active UC. In experimentally inflicted wounds, we found that neutrophils underwent NET formation in a PAD4-dependent manner hours after formation of primary blood clots, and remodelled clots to immunothrombi containing citrullinated histones, even in the absence of microbiota. PAD4-deficient mice experienced an exacerbated course of dextrane sodium sulfate-induced colitis with markedly increased rectal bleeding (96 % vs 10 %) as compared with controls. PAD4-deficient mice failed to remodel blood clots on mucosal wounds eliciting impaired healing. Thus, NET-associated immunothrombi are protective in acute colitis, while insufficient immunothrombosis is associated with rectal bleeding. CONCLUSION: Our findings uncover that neutrophils induce secondary immunothrombosis by PAD4-dependent mechanisms. Insufficient immunothrombosis may favour rectal bleeding in UC.

PICaSSO Histologic Remission Index (PHRI) in ulcerative colitis: development of a novel simplified histological score for monitoring mucosal healing and predicting clinical outcomes and its applicability in an artificial intelligence system.

Histological remission is evolving as an important treatment target in UC. We aimed to develop a simple histological index, aligned to endoscopy, correlated with clinical outcomes, and suited to apply to an artificial intelligence (AI) system to evaluate inflammatory activity. METHODS: Using a set of 614 biopsies from 307 patients with UC enrolled into a prospective multicentre study, we developed the Paddington International virtual ChromoendoScopy ScOre (PICaSSO) Histologic Remission Index (PHRI). Agreement with multiple other histological indices and validation for inter-reader reproducibility were assessed. Finally, to implement PHRI into a computer-aided diagnosis system, we trained and tested a novel deep learning strategy based on a CNN architecture to detect neutrophils, calculate PHRI and identify active from quiescent UC using a subset of 138 biopsies. RESULTS: PHRI is strongly correlated with endoscopic scores (Mayo Endoscopic Score and UC Endoscopic Index of Severity and PICaSSO) and with clinical outcomes (hospitalisation, colectomy and initiation or changes in medical therapy due to UC flare-up). A PHRI score of 1 could accurately stratify patients' risk of adverse outcomes (hospitalisation, colectomy and treatment optimisation due to flare-up) within 12 months. Our inter-reader agreement was high (intraclass correlation 0.84). Our preliminary AI algorithm differentiated active from quiescent UC with 78% sensitivity, 91.7% specificity and 86% accuracy. CONCLUSIONS: PHRI is a simple histological index in UC, and it exhibits the highest correlation with endoscopic activity and clinical outcomes. A PHRI-based AI system was accurate in predicting histological remission.