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Background & Aims: In contrast to mismatch repair deficient colorectal carcinoma (CRC), MMR proficient (pMMR) CRC does not respond to immune checkpoint blockade. We studied immune checkpoint stimulation via glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) on ex vivo functionality of human tumor-infiltrating lymphocytes (TIL) isolated from pMMR primary CRC and liver metastases (CRLM). Methods: Using lymphocytes from resected tumor, adjacent tissues, and peripheral blood mononuclear cells (PBMC) of 132 pMMR primary CRC or CRLM patients, we determined GITR expression and the in vitro T-cell agonistic activity of recombinant GITR ligation. Results: Here, we show that GITR was overexpressed on TIL when compared with other stimulatory immune checkpoints (4-1BB, OX40). Its expression was enhanced in TIL compared with PBMC and adjacent tissues. Among CD4+ TIL, GITR expression was primarily expressed by CD45RA- FoxP3hi activated regulatory T cells. Within CD8+ TIL, GITR was predominantly expressed on functionally exhausted and putative tumor-reactive CD103+ CD39+ TIL. Strikingly, recombinant GITRL reinvigorated ex vivo TIL responses by significantly enhancing CD4+ and CD8+ TIL numbers. Dual treatment with GITRL and nivolumab (anti-PD1) enhanced CD8+ TIL expansion compared with GITRL monotherapy. Moreover, GITRL/anti-PD1 dual therapy further improved anti-PD1-mediated reinvigoration of interferon gamma secretion by exhausted CD8 TIL from primary CRC. Conclusions: GITR is overexpressed on CD4+ and CD8+ TIL from pMMR CRC and CRLM. Agonistic targeting of GITR enhances ex vivo human TIL functionality and may therefore be a promising approach for novel monotherapy or combined immunotherapies in primary pMRR CRC and CRLM.

Background and study aims Accurate assessment of the lymph node (LN) status is crucial in resectable perihilar cholangiocarcinoma (pCCA) to prevent major surgery in patients with extraregional metastatic LNs (MLNs). This study investigates the added value of preoperative endoscopic ultrasound (EUS) with or without tissue acquisition (TA) for the detection of MLNs in patients with resectable pCCA. Patients and methods In this retrospective, multicenter cohort study, patients with potentially resectable pCCA who underwent EUS preoperatively between 2010–2020, were included. The clinical impact of EUS-TA was defined as the percentage of patients who did not undergo surgical resection due to MLNs found with EUS-TA. Findings of cross-sectional imaging were compared with EUS-TA findings and surgery. Results EUS was performed on 141 patients, of whom 107 (76 %) had suspicious LNs on cross-sectional imaging. Surgical exploration was prevented in 20 patients (14 %) because EUS-TA detected MLNs, of which 17 (85 %) were extraregional. Finally, 74 patients (52 %) underwent surgical exploration followed by complete resection in 40 (28 %). MLNs were identified at definitive pathology in 24 (33 %) patients, of which 9 (38 %) were extraregional and 15 (63 %) regional. Conclusions EUS-TA may be of value in patients with potentially resectable pCCA based on preoperative cross-sectional imaging, regardless of lymphadenopathy at cross-sectional imaging. A prospective study in which a comprehensive EUS investigation with LN assessment and EUS-TA of LNs is performed routinely should confirm this promise.

Autophagy is a cellular degradation mechanism, which is triggered by the bacterium Helicobacter pylori. A single nucleotide polymorphism (SNP) in the autophagy gene ATG16L1 (rs2241880, G-allele) has been shown to dysregulate autophagy and increase intestinal endoplasmic reticulum (ER) stress. Here, we investigate the role of this SNP in H. pylori-mediated gastric carcinogenesis and its molecular pathways. ATG16L1 rs2241880 was genotyped in subjects from different ethnic cohorts (Dutch and Australian) presenting with gastric (pre)malignant lesions of various severity. Expression of GRP78 (a marker for ER stress) was assessed in gastric tissues. The effect of ATG16L1 rs2241880 on H. pylori-mediated ER stress and pro-inflammatory cytokine induction was investigated in organoids and CRISPR/Cas9 modified cell lines. Development of gastric cancer was associated with the ATG16L1 rs2241880 G-allele. Intestinal metaplastic cells in gastric tissue of patients showed increased levels of ER-stress. In vitro models showed that H. pylori increases autophagy while reducing ER stress, which appeared partly mediated by the ATG16L1 rs2241880 genotype. H. pylori-induced IL-8 production was increased while TNF-α production was decreased, in cells homozygous for the G-allele. The ATG16L1 rs2241880 G-allele is associated with progression of gastric premalignant lesions and cancer. Modulation of H. pylori-induced ER stress pathways and pro-inflammatory mediators by ATG16L1 rs2441880 may underlie this increased risk.

Background & Aims: Antigen-specific immunotherapy is a promising strategy to treat HBV infection and hepatocellular carcinoma (HCC). To facilitate killing of malignant and/or infected hepatocytes, it is vital to know which T cell targets are presented by human leucocyte antigen (HLA)-I complexes on patient-derived hepatocytes. Here, we aimed to reveal the hepatocyte-specific HLA-I peptidome with emphasis on peptides derived from HBV proteins and tumour-associated antigens (TAA) to guide development of antigen-specific immunotherapy. Methods: Primary human hepatocytes were isolated with high purity from (HBV-infected) non-tumour and HCC tissues using a newly designed perfusion-free procedure. Hepatocyte-derived HLA-bound peptides were identified by unbiased mass spectrometry (MS), after which source proteins were subjected to Gene Ontology and pathway analysis. HBV antigen and TAA-derived HLA peptides were searched for using targeted MS, and a selection of peptides was tested for immunogenicity. Results: Using unbiased data-dependent acquisition (DDA), we acquired a high-quality HLA-I peptidome of 2 × 105 peptides that contained 8 HBV-derived peptides and 14 peptides from 8 known HCC-associated TAA that were exclusive to tumours. Of these, 3 HBV- and 12 TAA-derived HLA peptides were detected by targeted MS in the sample they were originally identified in by DDA. Moreover, 2 HBV- and 2 TAA-derived HLA peptides were detected in samples in which no identification was made using unbiased MS. Finally, immunogenicity was demonstrated for 5 HBV-derived and 3 TAA-derived peptides. Conclusions: We present a first HLA-I immunopeptidome of isolated primary human hepatocytes, devoid of immune cells. Identified HBV-derived and TAA-derived peptides directly aid development of antigen-specific immunotherapy for chronic HBV infection and HCC. The described methodology can also be applied to personalise immunotherapeutic treatment of liver diseases in general. Lay summary: Immunotherapy that aims to induce immune responses against a virus or tumour is a promising novel treatment option to treat chronic HBV infection and liver cancer. For the design of successful therapy, it is essential to know which fragments (i.e. peptides) of virus-derived and tumour-specific proteins are presented to the T cells of the immune system by diseased liver cells and are thus good targets for immunotherapy. Here, we have isolated liver cells from patients who have chronic HBV infection and/or liver cancer, analysed what peptides are presented by these cells, and assessed which peptides are able to drive immune responses.

Background & Aims: TIGIT is a co-inhibitory receptor, and its suitability as a target for cancer immunotherapy in HCC is unknown. PD1 blockade is clinically effective in about 20% of advanced HCC patients. Here we aim to determine whether co-blockade of TIGIT/PD1 has added value to restore functionality of HCC tumor-infiltrating T cells (TILs). Methods: Mononuclear leukocytes were isolated from tumors, paired tumor-free liver tissues (TFL) and peripheral blood of HCC patients, and used for flow cytometric phenotyping and functional assays. CD3/CD28 T-cell stimulation and antigen-specific assays were used to study the ex vivo effects of TIGIT/PD1 single or dual blockade on T-cell functions. Results: TIGIT was enriched, whereas its co-stimulatory counterpart CD226 was down-regulated on PD1high CD8+ TILs. PD1high TIGIT+ CD8+ TILs co-expressed exhaustion markers TIM3 and LAG3 and demonstrated higher TOX expression. Furthermore, this subset showed decreased capacity to produce IFN-γ and TNF-α. Expression of TIGIT-ligand CD155 was up-regulated on tumor cells compared with hepatocytes in TFL. Whereas single PD1 blockade preferentially enhanced ex vivo functions of CD8+ TILs from tumors with PD1high CD8+ TILs (high PD1 expressers), co-blockade of TIGIT and PD1 improved proliferation and cytokine production of CD8+ TILs from tumors enriched for PD1int CD8+ TILs (low PD1 expressers). Importantly, ex vivo co-blockade of TIGIT/PD1 improved proliferation, cytokine production, and cytotoxicity of CD8+ TILs compared with single PD1 blockade. Conclusions: Ex vivo, co-blockade of TIGIT/PD1 improves functionality of CD8+ TILs that do not respond to single PD1 blockade. Therefore co-blockade of TIGIT/PD1 could be a promising immune therapeutic strategy for HCC patients.

Patients with cholangiocarcinoma have poor survival since the majority of patients are diagnosed at a stage precluding surgical resection, due to locally irresectable tumors and/or metastases. Optimization of diagnostic strategies, with a principal role for tissue diagnosis, is essential to detect cancers at an earlier stage amenable to curative treatment. Current barriers for a tissue diagnosis include both insufficient tissue sampling and a difficult cyto- or histopathological assessment. During endoscopic retrograde cholangiopancreatography, optimal brush sampling includes obtaining more than one brush within an individual patient to increase its diagnostic value. Currently, no significant increase of the diagnostic accuracy for the new cytology brush devices aiming to enhance the cellularity of brushings versus standard biliary brush devices has been demonstrated. Peroral cholangioscopy with bile duct biopsies appears to be a valuable tool in the diagnostic work-up of indeterminate biliary strictures, and may overcome current technical difficulties of fluoroscopic-guided biopsies. Over the past years, molecular techniques to detect chromosomal instability, mutations and methylation profiling of tumors have revolutionized, and implementation of these techniques on biliary tissue during diagnostic work-up of biliary strictures may be awaited in the near future. Fluorescence in situ hybridization has already been implemented in routine diagnostic evaluation of biliary strictures in several centers. Next-generation sequencing is promising for standard diagnostic care in biliary strictures, and recent studies have shown adequate detection of prevalent genomic alterations in KRAS , TP53 , CDKN2A , SMAD4 , PIK3CA , and GNAS on biliary brush material. Detection of DNA methylation of tumor suppressor genes and microRNAs may evolve over the coming years to a valuable diagnostic tool for cholangiocarcinoma. This review summarizes optimal strategies for biliary tissue sampling during endoscopic retrograde cholangiopancreatography and focuses on the evolving molecular techniques on biliary tissue to improve the differentiation of benign and malignant biliary strictures.

Background and study aims Gastric cancer (GC) is usually preceded by premalignant gastric lesions (GPLs) such as gastric intestinal metaplasia (GIM). Information on risk factors associated with neoplastic progression of GIM are scarce. This study aimed to identify predictors for progression of GIM in areas with low GC incidence. Patients and methods The Progression and Regression of Precancerous Gastric Lesions (PROREGAL) study includes patients with GPL. Patients underwent at least two upper endoscopies with random biopsy sampling. Progression of GIM means an increase in severity according to OLGIM (operative link on gastric intestinal metaplasia) during follow-up (FU). Family history and lifestyle factors were determined through questionnaires. Serum Helicobacter pylori infection, pepsinogens (PG), gastrin-17 and GC-associated single nucleotide polymorphisms (SNPs) were determined. Cox regression was performed for risk analysis and a chi-squared test for analysis of single nucleotide polymorphisms. Results Three hundred and eight patients (median age at inclusion 61 years, interquartile range (IQR: 17; male 48.4 %; median FU 48 months, IQR: 24) were included. During FU, 116 patients (37.7 %) showed progression of IM and six patients (1.9 %) developed high-grade dysplasia or GC. The minor allele (C) on TLR4 (rs11536889) was inversely associated with progression of GIM (OR 0.6; 95 %CI 0.4–1.0). Family history (HR 1.5; 95 %CI 0.9–2.4) and smoking (HR 1.6; 95 %CI 0.9–2.7) showed trends towards progression of GIM. Alcohol use, body mass index, history of H. pylori infection, and serological markers were not associated with progression. Conclusions Family history and smoking appear to be related to an increased risk of GIM progression in low GC incidence countries. TLR4 (rs11536889) showed a significant inverse association, suggesting that genetic information may play a role in GIM progression.

Hepatocellular adenoma (HCA) is a rare, benign liver tumor. Discovery of this tumor is usually as an incidental finding, correlated with the use of oral contraceptives, or pregnancy. Treatment options have focused on conservative management for the straightforward, smaller lesions (5 cm) that pose a greater risk of hemorrhage or malignant progression. In recent years, a new molecular subclassification of HCA has been proposed, associated with characteristic morphological features and loss or increased expression of immunohistochemical markers. This subclassification could possibly provide considerable benefits in terms of patient stratification, and the selection of treatment options. In this review we discuss the decision-making processes and associated risk analyses that should be made based on lesion size, and subtype. The usefulness of this subclassification system in terms of the procedures instigated as part of the diagnostic work-up of a suspected HCA will be outlined, and suitable treatment schemes proposed.

Introduction: Emerging evidence suggests that long-term nucleos(t)ide analogue (NA) therapy can be ceased in a selective group of chronic hepatitis B (CHB). This is being gradually implemented in clinical practice. Case Presentation: A 68-year-old man known with a chronic hepatitis B e antigen-positive hepatitis B infection without signs of advanced liver fibrosis or cirrhosis was admitted with acute liver failure. Two months prior to his admission, he ceased his NA therapy. During the admission, NA therapy was restarted, but the liver function worsened. The patient was put on the high-urgency liver transplantation waiting list, and the next day, he was successfully transplanted. However, the patient died 17 days later due to hemorrhagic shock that resulted from intra-abdominal bleeding and acute pancreatitis. Conclusion: Current guidelines suggest that NA therapy can be discontinued in a selective group of CHB patients. However, these guidelines suggest different stopping and follow-up criteria. This case illustrates that NA withdrawal is not without risks and that these differences in recommendations may lead to inadequate management and eventually a fatal outcome.

Background & Aims: Individuals with obesity may develop intrapancreatic fat deposition (IPFD) and fatty pancreas disease (FPD). Whether this causes inflammation and fibrosis and leads to pancreatic dysfunction is less established than for liver damage in metabolic dysfunction-associated steatotic liver disease (MASLD). Moreover, the interrelations of FPD and MASLD are poorly understood. Therefore, we aimed to assess IPFD and fibro-inflammation in relation to pancreatic function and liver disease severity in individuals with MASLD. Methods: Seventy-six participants from the Amsterdam MASLD-MASH cohort (ANCHOR) study underwent liver biopsy and multiparametric MRI of the liver and pancreas, consisting of proton-density fat fraction sequences, T1 mapping and intravoxel incoherent motion diffusion-weighted imaging (IVIM-DWI). Results: The prevalence of FPD was 37.3%. There was a clear correlation between pancreatic T1 relaxation time, which indicates fibro-inflammation, and parameters of glycemic dysregulation, namely HbA1c (R = 0.59; p