Introduction

Recent genomic analysis of biliary tract carcinoma has revealed the distinct genetic features based on their primary sites and is expected to help develop primary site-specific treatments. However, the exact primary site of perihilar cholangiocarcinoma (phCCA), which is located between the intrahepatic and extrahepatic bile ducts, is virtually unknown. A precise diagnosis of the primary tumor site of phCCA is urgently necessary to select appropriate treatment and improve prognosis.

Aims & Methods

This study aims to identify the primary site of phCCA by molecular pathological analysis. We analyzed a total of 358 surgically treated invasive CCA operated at the National Cancer Centre Central Hospital Tokyo, Japan from 1990 to 2013. The primary site of the tumor was evaluated based on bile duct stenosis location on preoperative image findings, detailed macroscopic observation in the resected specimen and histological tumor localization using elastic fiber staining. Hilar CCA (hCCA) was defined as a tumor originating in the upper common, right or left hepatic duct without macroscopic mass formation in liver. The analysis set included 39 cases of mass-forming type intrahepatic CCA (iCCA-MF) and 9 cases of extrahepatic CCA (eCCA) in which both frozen and formalin-fixed paraffin-embedded specimens were available. The validation set included 60, 100, 85, 14, 36, and 15 cases of iCCA-MF, eCCA, hCCA, periductal infiltrating intrahepatic CCA (iCCA-PI), periductal infiltrating plus mass-forming intrahepatic CCA (iCCA-PI+Mass), and iCCA-MF with perihilar invasion, respectively. First, transcriptome analysis was performed using the analysis set to identify candidate genes differentially expressed between eCCA and iCCA-MF. Second, immunohistochemical staining of the candidate genes was performed on the analysis set to determine the evaluation criteria. Finally, immunostaining of the candidate markers was performed on the validation set to evaluate the expression levels of each CCA subtype and statistically analyze the comparison between hCCA and the other CCA subtypes.

Results

Candidate genes included Serpin Family A Member 1 (SEPRINA1), Claudin18 (CLDN18), and Mesothelin (MSLN) as positive cases, with their immunohistochemical staining evaluation criteria of ≥33%, ≥5%, and ≥1+ (positive intensity), respectively. The immunohistochemical expression of each three molecules in the analysis set between eCCA and iCCA-MF revealed SERPINA1 at 0% vs. 82% (P < 0.01), CLDN18 at 100% vs. 41% (P < 0.01), and MSLN at 100% vs. 48% (P < 0.01). Similarly, a statistically significant difference was observed in the expression of each of the three molecules in the validation set. However, no significant difference was found in the immunohistochemical expression of the three molecules between eCCA and hCCA. Conversely, SERPINA1 expression in iCCA-MF cases with perihilar invasion was significantly different from that in cases of hCCA with microscopic hepatic parenchyma invasion, but not significantly different from that in cases of iCCA-PI+Mass. Furthermore, clustering analysis of the combination of three molecule expressions revealed that CCA was divided into three subgroups, consisting of iCCA-MF, iCCA-PI+Mass and iCCA-PI, and hCCA and eCCA.

Conclusion

We demonstrated molecular pathologically that the primary tumor site of phCCA can be identified and clinicopathologically classified in detail. In particular, we succeeded in showing that hCCA is classified into the same group as eCCA.

Introduction

The use of 3D cell culture models, including spheroids and organoids, has proven to be a valuable method to approximate in vivo conditions in basic research. Our recent work on the pathogenesis of spontaneous bacterial peritonitis (SBP) – a life-threatening complication of liver cirrhosis with a 1-year-mortality of 66% – shows that bacteria induce the degradation of important cell-cell contact proteins upon direct contact via various processes. The resulting destabilization of the epithelial barrier enables bacterial translocation and thus represents a mechanism in the development of SBP (Haderer et al., Gut 2022). To evaluate these processes under more physiological conditions, we investigate bacterial-induced effects on cell-cell contact proteins in a 3D spheroid model and subsequently in a patient-based organoid model.

Aims & Methods

To establish the method and create a reference, spheroids were generated from colon carcinoma cell line HCT116 with a concentration of 70,000 cells/ml. Spheroid development and formation of cell-cell contacts were assessed by fluorescence microscopy. Subsequently, bacterial-induced effects on the tight junction proteins Occludin, Claudin4 and Zonulin1 as well as on the adherens junction protein E-Cadherin were investigated after up to 12 hours of bacterial co-culture using qPCR, fluorescence microscopy and Western blot analysis. Bacterial co-culture was performed with the reference strain Escherichia coli (E.coli) O6:Hnt and the E. coli strain Ont:H41, which was isolated from the ascites of a patient with SBP.
In addition, organoids were generated from intestinal biopsies of patients with liver cirrhosis and co-cultured with both bacterial strains. Bacterial-induced effects on cell-cell-contact proteins were investigated using Western blot analysis.

Results

HCT116 cells formed formed spheroids composed of viable cells with well-defined cell-cell contacts. Co-culture with E. coli for up to 12 hours led to a significant degradation of Occludin (up to 60% reduction), E-Cadherin (up to 85% reduction) and Zonulin1 (up to 95% reduction), whereas Claudin4 levels remained mostly constant. Co-culture with heat-inactivated bacteria did not affect cell-cell contacts. The intensity of these effects was dependent on the respective protein, the bacterial strain used, as well as the multiplicity of infection (MOI) and the duration of the co-culture. Degradation of Occludin could also be visualized by fluorescence microscopy.
We were then able to transfer this experimental approach to an organoid setting. Bacterial-induced degradation of the epithelial barrier was confirmed in organoids, showing a clearly visible reduction of E-Cadherin, Occludin and Zonulin1 after 8 hours of bacterial co-culture. The intensity of the effects varied depending on the bacterial strain, with the patient strain showing a slightly higher level of aggressiveness.

Conclusion

Understanding the pathophysiological changes of the intestinal epithelial barrier in patients with liver cirrhosis is essential for advancing therapeutic strategies for SBP. In this context, a representative ex vivo model is crucial to draw reliable conclusions. Our work shows that relevant mechanisms of SBP, such as bacterial-induced degradation of cell-cell contacts, can be successfully modeled in spheroids and organoids. Thus, our study provides an approach for further approximation to in vivo conditions – bridging the gap between basic research and clinical utility.

References

Haderer M, Neubert P, Rinner E, Scholtis A, Broncy L, Gschwendtner H, Kandulski A, Pavel V, Mehrl A, Brochhausen C, Schlosser S, Gülow K, Kunst C, Müller M. Novel pathomechanism for spontaneous bacterial peritonitis: disruption of cell junctions by cellular and bacterial proteases. Gut. 2022 Mar;71(3):580-592. doi: 10.1136/gutjnl-2020-321663 . Epub 2021 Mar 11. PMID: 33707230 ; PMCID: PMC8862089.

Introduction

Alterations in body composition and metabolic factors may serve as a potential biomarker for early-detection of pancreatic ductal adenocarcinoma (PDAC).

Aims & Methods

This study aimed to capture the longitudinal changes occurring in body composition and metabolic factors preceding a diagnosis of PDAC. All patients (≥18 years) diagnosed with PDAC between 2000 and 2021 were identified. We collected data on demographics, comorbidities, PDAC characteristics, all abdominal CT scans and blood-based markers up to 3 years prior to date of diagnosis (index date). We applied a fully-automated abdominal segmentation algorithm previously developed by our group (Weston et al. Radiology 2019 [1]) for three-dimensional quantification of body composition on CT scans. Longitudinal trends of body composition and laboratory markers prior to a diagnosis of PDAC were estimated by linear mixed models and visualized using spline regression.

Results

We included 1,825 PDAC patients in body composition analysis, of which 581 (31.8%) had ≥2 pre-diagnostic CT exams. For analysis of longitudinal trends of blood-based markers, 4,846 patients were included. Mean age at diagnosis was 68.4 (SD 11.3) years, 43.6% were female and the mean BMI was 27.0 (SD 5.5). The distribution of PDAC stage was as follows: 12.5% stage I, 32.6% stage II, 19.7% stage III, and 35.2% stage IV. Prior to a PDAC diagnosis, we observed significant decreases in visceral fat volume, subcutaneous fat volume, bone volume, and a variety of blood markers including low-density lipoprotein and hemoglobin (Table 1). Concurrently, patients developed significant increases in HbA1c, fasting blood glucose, high-density lipoprotein and white blood cells prior to a clinical PDAC diagnosis. Most body composition and metabolic markers showed a linear decreasing or increasing trend over time to diagnosis of PDAC.

Table 1. Trends of body composition and laboratory markers over time prior to a diagnosis of PDAC estimated by linear mixed models.

Conclusion

This study identified significant alterations in a variety of soft tissue and metabolic markers that occur in the development of PDAC. These metabolic changes may become apparent several years before diagnosis, which may serve as a significant window of opportunity for early detection.

References

1. Weston AD, Korfiatis P, Kline TL, Philbrick KA, Kostandy P, Sakinis T, Sugimoto M, Takahashi N, Erickson BJ. Automated Abdominal Segmentation of CT Scans for Body Composition Analysis Using Deep Learning. Radiology. 2019 Mar;290(3):669-679. doi: 10.1148/radiol.2018181432. Epub 2018 Dec 11. PMID: 30526356.

Disclosure

On behalf of all authors, I declare no conflicts of interest.

Introduction

Small bowel adenocarcinoma (SBA) are rare, but their incidence is increasing. Early diagnosis of SBA remains elusive, so that about one-third of patients are initially diagnosed with distant metastases. In clinical practice, regimens used for SBA parallel those commonly administered in the adjuvant setting for colorectal cancer, owing to its anatomic proximity. However, genomic profiling of SBA has been shown to be distinct from colorectal cancer through comprehensive analysis of genomic alterations. Therefore, it is vital to establish a distinct chemotherapy regimen for SBA based on its molecular pathological characteristics. Claudin-18 plays a key role in constructing tight junctions and altered claudin-18 expression has been documented in various human malignancies. On the basis of this, both monoclonal antibodies and claudin-18.2-specific chimeric antigen receptor engineered T-cells have been recently developed and employed in several clinical trials, showing promising results in the treatment of advanced gastroesophageal and pancreatobiliary tract cancers. However, little is known about the clinicopathological significance of claudin-18 expression in SBA.

Aims & Methods

To explore the localization of claudin-18 in human SBA tissue and draw comparisons and determine the relationship between their expression and clinicopathological factors, and genomic alterations.
We examined 50 surgical resections from patients with primary SBA for immunohistochemical analyses of claudin-18, CK7, CK20, CD10, CDX2, MUC2, MUC5AC, and MUC6 expression. DNA mismatch repair (MMR) status was defined by immunostaining for all MMR proteins; MLH1, MSH2, MLH6, and PMS2. Both MUC 2 and CD10 are markers of intestinal type tumors, whereas MUC5AC and MUC6 are markers of gastric type tumors. Tissue samples showing both gastric and intestinal phenotypes were classified as gastrointestinal type tumors, whereas those showing neither gastric nor intestinal phenotype expression were classified as null type tumors. In addition, genomic profiling was performed on 31 patients using targeted next-generation sequencing.

Results

Claudin-18 expression was observed in 46% of SBA patients. Claudin-18 expression was positively associated with cytokeratin 7 (P=0.003) and MUC5AC (P=0.020) expression, whereas it was negatively associated with MUC2 (P=0.010), CD10 (P=0.014), and CDX2 (P=0.003) expression. Tumor mucin phenotypes included gastric type (n=9), intestinal type (n=20), gastrointestinal type (n=17) and null type (n=4). Claudin-18 was more frequently expressed in gastric type (100%) than in intestinal type (30%) phenotypes (P=0.001). GNAS mutations (N=4) were found in only claudin-18 positive SBA patients, although the difference was not significant. Claudin-18 expression did not correlate with patient age at diagnosis, patient gender, site, histological type, depth of invasion, lymph node metastasis, distal metastasis, TNM stage, and MMR status. However, claudin-18 expression correlated with worse progression free survival.

Conclusion

Claudin-18 expression is associated with gastric immunophenotype and with worse progression free survival. These findings suggest that claudin-18 may be a potential therapeutic target in certain SBA cases.

Introduction

A low incidence of eosinophilic esophagitis (EoE) in children in the North Denmark Region (NDR) were measured in 2007-2017. Few of the children diagnosed before 2017 were treated to remission suggesting a lack of awareness. While there currently are no guidelines for treating EoE in Denmark, a new English guideline was published in 2022 renewing focus on the disease.

Aims & Methods

The aim of this study was to measure the difference of current Danish clinical practice for treatment and follow-up of EoE children in the NDR with the new English guideline from the British Society of Gastroenterology (BSG) and the British Society of Pediatric Gastroenterology, Hepatology and Nutrition (BSPGHAN). This retrospective, register-based DanEoE cohort study included 31 children diagnosed with EoE between 2007–2021 in NDR. Medical records were reviewed and information about treatment and follow-up were collected.

Results

In 32% of the children with EoE in the NDR, first-line treatment initiated corresponded with the recommendations in the new English guideline. One in 6 children were never started on any treatment even though treatment always is recommended. Symptomatic follow-up was completed in 77% (24/31) of children and 85% (11/13) of the children after 2017. Histologic follow-up within 12 weeks was completed in 13% (4/31) of the children and 15% (2/13) of the children after 2017. Combined symptomatic and histologic remission after first-line treatment was achieved in 6% (2/31).

Conclusion

In Denmark focus on improving EoE treatment and follow-up for children is needed, as there is a significant difference between current clinical practice and the recommendations in the new English guideline.

References

Hollænder M, Terkelsen JH, Kramme F, Bredal K, Kragholm K, Dalby K, et al. The incidence of eosinophilic oesophagitis in 2007-2017 among children in North Denmark Region is lower than expected. BMC Pediatr. 2022 Apr;22(1):183.
Dhar A, Haboubi HN, Attwood SE, Auth MKH, Dunn JM, Sweis R, et al. British Society of Gastroenterology (BSG) and British Society of Paediatric Gastroenterology, Hepatology and Nutrition (BSPGHAN) joint consensus guidelines on the diagnosis and management of eosinophilic oesophagitis in children and adults. Gut. 2022 Aug;71(8):1459–87.

Introduction

As a chronic condition, eosinophilic oesophagitis (EoE) requires lifelong therapeutic management and multidisciplinary care. Reports suggest clinicians managing EoE have gaps in knowledge and competence, which may lead to suboptimal patient outcomes. We aimed to assess whether a purpose-specific curriculum comprising a series of continuing medical education (CME) activities addressed these gaps.

Aims & Methods

Four faculty-led, CME-accredited activities on EoE were hosted on two free-to-access online medical education websites. Each activity provided 30–60 minutes of education in short videos and addressed learning objectives based on a review of published literature and expert insights. Educational outcomes were assessed using Moore’s expanded outcomes framework Levels 1–4 (participation, satisfaction, knowledge, and competence, respectively). Intent to change practice and need for further education were also assessed. Level 1 data were captured using Google Analytics; Level 2–4 data were captured using pre- and post-activity questionnaires administered by an independent third party and analysed by the authors. Across the curriculum 21,032 participants engaged with the content, with a sample size of 800 available for the Level 2–4 analysis; the margin of error was calculated as ~3% based on a standard confidence interval of 95%.

Results

Within 12 weeks of launch, a mean of 5,258 participants engaged with each curriculum activity (Moore’s Level 1). Mean satisfaction (Moore’s Level 2) was high (87.3%) across the curriculum. From baseline to follow-up, significant average increases in declarative knowledge (24%, p=0.012), procedural knowledge (18%, p<0.001), and competence (16%, p=0.004) were observed (Moore’s Levels 3a, 3b, and 4, respectively) (Table). Gastroenterologists had the highest proportion of correct answers at follow-up across all activities (86–95%). Allergists/immunologists had the largest increase in knowledge from baseline to follow-up (51%, activity 1); the smallest increase was seen for gastroenterologists (7%, activity 2). For all specialities, the lowest proportion of correct answers at follow-up was for questions on pathophysiology/causes of EoE (Table). Overall, 64% of learners would change their practice following participation in ≥1 curriculum activity and ~20% indicated further education would be beneficial.

Conclusion

This expert-led, CME curriculum led to significant improvements in clinician knowledge and competence surrounding the latest research and best practices in the pathophysiology, diagnosis, and management of EoE, which may lead to improvements in patient care and outcomes. Findings support further development of online, multiformat educational activities to address clinical knowledge and competence gaps. Future programs should focus on the underlying pathophysiology of EoE and how this may inform practice.

References

None

Disclosure

Stuart Carr discloses: Consulting fees/advisory board: Sanofi. Honoraria (speakers bureau): Sanofi, Biologix.
Evan S. Dellon discloses: Research funding: Adare/Ellodi, Allakos, Arena, AstraZeneca, GSK, Meritage, Miraca, Nutricia, Celgene/Receptos/BMS, Regeneron, Revolo, Shire/Takeda. Consultant: Abbott, Abbvie, Adare/ Ellodi, Aimmune, Akesobio, Alfasigma, ALK, Allakos, Amgen, Aqilion, Arena/Pfizer, Aslan, AstraZeneca, Avir, Biorasi, Calypso, Celgene/Receptos/BMS, Celldex, Eli Lilly, EsoCap, Eupraxia, Ferring, GSK, Gossamer Bio, Holoclara, Invea, Landos, LucidDx, Morphic, Nexstone Immunology, Nutricia, Parexel/Calyx, Phathom, Regeneron, Revolo, Robarts/Alimentiv, Salix, Sanofi, Shire/Takeda, Target RWE, Upstream Bio. Educational grant: Allakos, Holoclara, Invea
Nirmala Gonsalves discloses: Consultant: Allakos, Astra-Zeneca, Regeneron-Sanofi, BMS, Knopp. Speaking: Regeneron-Sanofi. Royalties: Up To Date.
Salvatore Oliva discloses: Consulting fees from Sanofi.
Jonathan M. Spergel discloses: Consultant: Regeneron and Sanofi-Genzyme, Novartis, Allakos. Grants/Research Support: NIH, FARE, Regeneron, Novartis, Sanofi, Celgene. Speaker’s Bureau: Medscape.
Mário C. Vieira: No conflicts of interest associated with the submitted work to declare.
Katie Bickford, Kathy Day, Alex Noble, Anne Nunn and Alison Scott: No financial relationships to disclose.