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Background & Aims Little is known about the composition of intrahepatic immune cells and their contribution to the pathogenesis of primary sclerosing cholangitis (PSC). Herein, we aimed to create an atlas of intrahepatic T cells and thereby perform an in-depth characterization of T cells in inflamed human liver. Methods Different single-cell RNA sequencing methods were combined with in silico analyses on intrahepatic and peripheral T cells from patients with PSC (n = 11) and healthy donors (HDs, n = 4). Multi-parameter flow cytometry and functional in vitro experiments were conducted on samples from patients with PSC (n = 24), controls with other liver diseases and HDs. Results We identified a population of intrahepatic naive-like CD4+ T cells, which was present in all liver diseases tested, but particularly expanded in PSC. This population had a transcriptome and T cell receptor repertoire similar to circulating naive T cells but expressed a set of genes associated with tissue residency. Their periductal location supported the concept of tissue-resident naive-like T cells in livers of patients with PSC. Trajectory inference suggested that these cells had the developmental propensity to acquire a T helper 17 (TH17) polarization state. Functional and chromatin accessibility experiments revealed that circulating naive T cells in patients with PSC were predisposed to polarize towards TH17 cells. Conclusion We report the first atlas of intrahepatic T cells in PSC, which led to the identification of a previously unrecognized population of tissue-resident naive-like T cells in the inflamed human liver and to the finding that naive CD4+ T cells in PSC harbour the propensity to develop into TH17 cells. Lay summary The composition of intrahepatic immune cells in primary sclerosing cholangitis (PSC) and their contribution to disease pathogenesis is widely unknown. We analysed intrahepatic T cells and identified a previously uncharacterized population of liver-resident CD4+ T cells which are expanded in the livers of patients with PSC compared to healthy liver tissue and other liver diseases. These cells are likely to contribute to the pathogenesis of PSC and could be targeted in novel therapeutic approaches., Graphical abstract, Highlights • First single-cell atlas of intrahepatic T cell landscape in PSC. • Identification of tissue-resident naive-like CD4+ T cells in human liver which are expanded in livers of patients with PSC. • Trajectory inference suggested a developmental propensity of these cells to acquire a TH17 polarization state. • Functional experiments, using circulating naive CD4+ T cells, support the inferred propensity to acquire a TH17 polarization state. • Chromatin accessibility studies revealed imprinting of naive CD4+ T cells towards effector function in PSC.

Biliary stenoses represent a differential diagnostic challenge. Diagnostic methods to clarify the underlying dignity are often invasive, and provide high specificity beside insufficient sensitivity. In many cases, an accurate diagnosis is only possible over time, and therefore limits curative treatment options. This article provides an overview of the diagnostic challenges and treatment options for unclear biliary stenosis.

Introduction: Cancer-associated fibroblasts (CAF) have been identified as relevant contributors to cancer progression and drug resistance in many tumors. Although neuroendocrine tumors (NET) are often associated with a strong stromal reaction, no study has addressed whether CAF are involved in progression and therapeutic resistance in NET. The aim of this study was to characterize the role of CAF in NET. Methods: We established primary CAF cultures derived from NET liver metastases to study the effect on NET cell lines NT-3 and BON. Immunohistochemistry was performed on tissue sections of primary and metastatic NET tissue. Results: Immunohistochemistry identified CAF dispersed in between tumor cells and within fibrotic bands separating tumor cell clusters in NET. Stimulating NET cells with CAF decreased expression of SSTR2 and chromogranin A and induced expression of CXCR4. CAF induced a 2.3-fold increase in proliferation and completely reversed the response to everolimus in NT-3 cells. We identified STAT3 as the main signaling pathway induced by CAF. STAT3 targeting by small interfering RNA knockdown and inhibitors prevented CAF-induced proliferation and restored everolimus responsiveness. STAT3 activation in NET tissue was associated with decreased chromogranin A expression, increased Ki-67 index, and decreased 5-year overall and progression-free survival. CAF directly influence proliferation and therapeutic response in NET cells. Conclusion: Identifying STAT3 as the contributing pathway of this so far neglected tumor-stroma interaction has the potential to become a new therapeutic target to halt tumor growth and to restore therapeutic responsiveness in NET.

Background Following endoscopic resection of early-stage Barrett’s esophageal adenocarcinoma (BEA), further oncologic management then fundamentally relies upon the accurate assessment of histopathologic risk criteria, which requires there to be sufficient amounts of submucosal tissue in the resection specimens. Methods In 1685 digitized tissue sections from endoscopic mucosal resection (EMR) or endoscopic submucosal dissection (ESD) performed for 76 early BEA cases from three experienced centers, the submucosal thickness was determined, using software developed in-house. Neoplastic lesions were manually annotated. Results No submucosa was seen in about a third of the entire resection area (mean 33.8 % [SD 17.2 %]), as well as underneath cancers (33.3 % [28.3 %]), with similar results for both resection methods and with respect to submucosal thickness. ESD results showed a greater variability between centers than EMR. In T1b cancers, a higher rate of submucosal defects tended to correlate with R1 resections. Conclusion The absence of submucosa underneath about one third of the tissue of endoscopically resected BEAs should be improved. Results were more center-dependent for ESD than for EMR. Submucosal defects can potentially serve as a parameter for standardized reports.

Background The translational interest in the intratumoral heterogeneity of hepatocellular carcinoma (HCC) has been increasing. The dismal prognosis of this pathology is linked to the features of the HCC harbouring cancer stem cells (CSC), represented by EpCAM-expression. However, the extent of the impact of intratumoral distribution of CSC-features, both on the recurrence after curative resection and on clinical outcome, remains unknown. To address this, we investigated the spatial heterogeneity of CSC-features with the aim of identifying the unique HCC patient subgroups amenable to adjuvant treatment. Methods We designed a tissue microarray (TMA) from patients who had received liver resection between 2011 and 2017. Tumor specimens were sampled at multiple locations (n = 3–8). EpCAM-positivity was assessed for intensity and proportion by applying a score dividing three groups: (i) negative (E−/−); (ii) heterogeneous (E−/+); and (iii) homogeneous (E+/+). The groups were further analysed with regard to time-to-recurrence (TTR) and recurrence-free-survival (RFS). Results We included 314 tumor spots from 69 patients (76.8% male, median age 66, liver cirrhosis/fibrosis 75.8%). The risk factors were alcohol abuse (26.2%), NASH (13.1%), HBV (15.5%), HCV (17.9%) and others (27.4%), representative of a typical Western cohort. E+/+ patients experienced significantly shorter TTR and RFS compared to E+/− and E−/− patients (TTR 5 vs. 19 months, p = 0.022; RFS 5 vs. 14 vs. 21 months, p = 0.016). Only homogeneous EpCAM-positivity correlated with higher AFP levels (> 400 ng/ml, p = 0.031). Conclusions Spatial heterogeneity of EpCAM-expression was markedly present in the cohort. Of note, only homogeneous EpCAM-expression correlated significantly with early recurrence, whereas heterogeneous EpCAM-expression was associated with clinical endpoints comparable to EpCAM-negativity. We identified a unique HCC subtype associated with a high risk of tumor recurrence.

Backgrounds & Aims: Intratumoural heterogeneity of hepatocellular carcinoma (HCC) is of increasing translational interest. Dismal prognosis is frequently linked to HCC harbouring cancer stem cell (CSC)-features, represented by EpCAM-expression. However, to what extent intratumoural distribution of CSC-features impacts on recurrence after curative resection remains unknown. Hence, we aimed to investigate the spatial heterogeneity of CSC-features and its impact on clinical outcome, identifying high-risk patients amenable to adjuvant treatment.Methods: We designed a tissue microarray (TMA) from patients, who received liver resection between 2011 and 2017. Tumour specimens were sampled at multiple locations (n=3-8). EpCAM-positivity was assessed for intensity and proportion by applying a score dividing three groups: negative (E-/-), heterogeneous-positive (E-/+), homogeneous-positive (E+/+). The groups were further analysed with respect to time-to-recurrence (TTR) and recurrence-free-survival (RFS).Results: We included 341 tumour spots from 75 patients (77% male, median age 66 years, liver cirrhosis/fibrosis 74.6%). Risk factors were alcohol abuse in 23.9%, NASH 16.3%, HBV 14.1%, HCV 17.4% and others 28.3%, representing a typical Western cohort. E+/+ patients experienced a significantly shorter TTR and RFS compared to E+/- (and E-/-) patients (TTR 5 vs. 19 months, p=0.017; RFS 5 vs. 14 vs. 18 months, p=0.016). Only homogeneous EpCAM-positivity correlated with higher AFP levels (>400 ng/ml, p=0.024).Conclusions: Spatial heterogeneity of EpCAM-expression was markedly present. Only homogeneously positive EpCAM-expression correlated significantly with early recurrence, whereas heterogeneous EpCAM-expression was associated with clinical endpoints comparable to EpCAM-negativity. Similar to colorectal cancer, high or low risk features for recurrence could be decisive for adjuvant treatment.

Background and aimsEndoscopic 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.MethodsThis 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.ResultsCancer 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.ConclusionsTumour 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.

Biliary stenoses represent a differential diagnostic challenge. Diagnostic methods to clarify the underlying dignity are often invasive, and provide high specificity beside insufficient sensitivity. In many cases, an accurate diagnosis is only possible over time, and therefore limits curative treatment options. This article provides an overview of the diagnostic challenges and treatment options for unclear biliary stenosis.

Histopathologic diagnosis relies on simultaneous integration of information from a broad range of scales, ranging from nuclear aberrations ($\approx \mathcal{O}(0.1{\mu m})$) through cellular structures ($\approx \mathcal{O}(10{\mu m})$) to the global tissue architecture ($\gtrapprox \mathcal{O}(1{mm})$). To explicitly mimic how human pathologists combine multi-scale information, we introduce a family of multi-encoder FCNs with deep fusion. We present a simple block for merging model paths with differing spatial scales in a spatial relationship-preserving fashion, which can readily be included in standard encoder-decoder networks. Additionally, a context classification gate block is proposed as an alternative for the incorporation of global context. Our experiments were performed on three publicly available whole-slide images of recent challenges (PAIP 2019, BACH 2020, CAMELYON 2016). The multi-scale architectures consistently outperformed the baseline single-scale U-Nets by a large margin. They benefit from local as well as global context and particularly a combination of both. If feature maps from different scales are fused, doing so in a manner preserving spatial relationships was found to be beneficial. Deep guidance by a context classification loss appeared to improve model training at low computational costs. All multi-scale models had a reduced GPU memory footprint compared to ensembles of individual U-Nets trained on different image scales. Additional path fusions were shown to be possible at low computational cost, opening up possibilities for further, systematic and task-specific architecture optimization. The findings demonstrate the potential of the presented family of human-inspired, end-to-end trainable, multi-scale multi-encoder FCNs to improve deep histopathologic diagnosis by extensive integration of largely different spatial scales., Comment: Accepted for Medical Image Analysis

This chapter presents information about three types of study designs commonly used for liver disease research. Each section provides a set of terms used when presenting research using the given design, an overview of the design, key aspects of the methods used, strengths and limits of the design, and a clinical vignette which draws from relevant research on a liver disease and highlights key aspects of the design.

HSD3B2 plays a crucial role in steroid hormone biosynthesis and is thus of particular interest in hormone dependent tumors such as prostate cancer. To clarify the clinical relevance of HSD3B2 expression in prostate cancer, we analyzed HSD3B2 protein expression by immunohistochemistry on our preexisting tissue microarray with 12.247 annotated cancers. Compared with normal tissue cytoplasmic HSD3B2 staining was stronger in prostate cancers. In 9371 interpretable cancers, HSD3B2 expression was found in 95.5% of cancers and was considered weak in 29.9%, moderate in 40.7% and strong in 24.9%. HSD3B2 up regulation was linked to advanced pathological tumor stage (pT), high Gleason grade, elevated preoperative PSA levels (p 0.0001 each), lymph node metastasis (p = 0.0019), accelerated cell proliferation (p 0.0001), androgen receptor (AR) expression (p 0.0001), and early biochemical recurrence (p 0.0001). HSD3B2 up regulation was only marginally more frequent in ERG positive (98%) than in ERG negative cancers (94%; p 0.0001) and was strongly linked to deletions of 5q and 6q (p 0.0001 each). Multivariate analyses showed that the prognostic impact of HSD3B2 expression was independent of established preoperative, but not of postoperative prognostic parameters. In summary, the results of our study demonstrate that HSD3B2 is strongly up regulated in a fraction of prostate cancers that are characterized by increased AR signaling, adverse tumor phenotype and early biochemical recurrence.

The controlled attenuation parameter (CAP) measured by transient elastography allows for the noninvasive assessment of hepatic steatosis. However, discrepant results between CAP values and histological evaluation have been reported in particular with high CAP values. We therefore investigated the diagnostic validity of high CAP measurements.Forty patients with liver disease and CAP measurements 300 dB/m that underwent ultrasound-guided or minilaparoscopic liver biopsy were retrospectively enrolled. CAP values were compared with the respective histological and macroscopic evaluation and correlated with clinical parameters.CAP values 300 dB/m had an 87.5 % specificity for detection of hepatic steatosis but failed to discriminate between steatosis grade S1 - S3. Discordant results, defined as a discrepancy of at least 2 steatosis grades between transient elastography and liver biopsy, were observed in 40 % of cases. The interquartile range (IQR) of CAP was confirmed as a predictor of discrepant findings. Macroscopic evaluation as part of minilaparoscopy detected hepatic steatosis in 74 % of patients with histological grade S2 - S3 in contrast to only 10 % classified as histological grade S0 - S1.High CAP measurements need to be interpreted with care and with regard to clinical parameters, in particular when high IQR values are registered.ZIEL: Der durch transiente Elastografie gemessene Controlled Attenuation Parameter (CAP) ermöglicht die nicht-invasive Beurteilung der Steatosis hepatis. Insbesondere im Falle hoher CAP-Werte wurden jedoch abweichende Ergebnisse zwischen CAP-Werten und histologischer Untersuchung berichtet. Ziel dieser Studie ist es, die diagnostische Gültigkeit hoher CAP-Messungen zu untersuchen.40 Patienten mit Lebererkrankung und CAP-Messungen 300 dB/m sowie sonografisch gesteuerter oder minilaparoskopischer Leberbiopsie wurden retrospektiv eingeschlossen. Die CAP-Werte wurden mit den jeweiligen histologischen und makroskopischen Beurteilungen verglichen sowie mit klinischen Parametern korreliert.CAP-Werte 300 dB/m wiesen eine Spezifität von 87,5 % für den Nachweis einer Steatosis hepatis auf, konnten jedoch zwischen den Steatosegraden S1 – S3 nicht unterscheiden. Diskordante Ergebnisse, definiert als eine Diskrepanz von mindestens zwei Steatosegraden zwischen transienter Elastografie und Leberbiopsie, wurden in 40 % der Fälle beobachtet. Der Interquartilsabstand der CAP-Messung wurde als Prädiktor für diskrepante Befunde bestätigt. Die makroskopische Beurteilung im Rahmen einer Minilaparoskopie lieferte in 74 % der Patienten mit histologischer Steatosis S2 – S3 vs. 10 % der Patienten mit histologischer Steatosis S0 – S1 Hinweise auf eine vorliegende Steatosis hepatis.Hohe CAP-Messungen müssen insbesondere im Falle ebenfalls erhöhter IQR-Werte sorgfältig und in Zusammenschau mit den klinischen Parametern bewertet werden.

Rationale: Ventricular arrhythmias remain the leading cause of death in patients suffering myocardial ischemia. Myeloperoxidase, a heme enzyme released by polymorphonuclear neutrophils, accumulates within ischemic myocardium and has been linked to adverse left ventricular remodeling. Objective: To reveal the role of myeloperoxidase for the development of ventricular arrhythmias. Methods and Results: In different murine models of myocardial ischemia, myeloperoxidase deficiency profoundly decreased vulnerability for ventricular tachycardia on programmed right ventricular and burst stimulation and spontaneously as assessed by ECG telemetry after isoproterenol injection. Experiments using CD11b/CD18 integrin–deficient (CD11b −/− ) mice and intravenous myeloperoxidase infusion revealed that neutrophil infiltration is a prerequisite for myocardial myeloperoxidase accumulation. Ventricles from myeloperoxidase-deficient (Mpo −/− ) mice showed less pronounced slowing and decreased heterogeneity of electric conduction in the peri-infarct zone than wild-type mice. Expression of the redox-sensitive gap junctional protein Cx43 (Connexin 43) was reduced in the peri-infarct area of wild-type compared with Mpo −/− mice. In isolated wild-type cardiomyocytes, Cx43 protein content decreased on myeloperoxidase/H 2 O 2 incubation. Mapping of induced pluripotent stem cell–derived cardiomyocyte networks and in vivo investigations linked Cx43 breakdown to myeloperoxidase-dependent activation of matrix metalloproteinase 7. Moreover, Mpo −/− mice showed decreased ventricular postischemic fibrosis reflecting reduced accumulation of myofibroblasts. Ex vivo, myeloperoxidase was demonstrated to induce fibroblast-to-myofibroblast transdifferentiation by activation of p38 mitogen-activated protein kinases resulting in upregulated collagen generation. In support of our experimental findings, baseline myeloperoxidase plasma levels were independently associated with a history of ventricular arrhythmias, sudden cardiac death, or implantable cardioverter–defibrillator implantation in a cohort of 2622 stable patients with an ejection fraction >35% undergoing elective diagnostic cardiac evaluation. Conclusions: Myeloperoxidase emerges as a crucial mediator of postischemic myocardial remodeling and may evolve as a novel pharmacological target for secondary disease prevention after myocardial ischemia.