LEMD2-associated progeroid syndrome: Expanding the phenotype of the nuclear envelopathy caused by a defect in LEMD2 gene.

Nuclear envelopathies are rare genetic diseases that compromise the integrity of the nuclear envelope. Patients with a defect in LEM domain nuclear envelope protein 2 (LEMD2) leading to LEMD2-associated progeroid syndrome are exceedingly scarce in number, yet they exhibit shared clinical features including skeletal abnormalities and a prematurely-aged appearance. Our study broadens the understanding of LEMD2-associated progeroid syndrome by detailing its phenotypic and molecular characteristics in the first female and fourth reported case, highlighting a distinct impact on metabolic functions. The patient's history revealed growth delay, facial and skeletal abnormalities, and recurrent abdominal pain crises caused by hepatomegaly. Comparisons with the previously documented cases emphasized similarities in skeletal and facial features while showcasing unique variations, notably in cardiac and hepatic manifestations. In vitro experiments conducted on patient-derived peripheral blood and urinary epithelial cells and LEMD2-downregulated HepG2 cells confirmed abnormalities in the structure of the nuclear envelope in all three tissue-types. Overall, our work offers a comprehensive profile of a patient with LEMD2-related syndrome, emphasizing the hepatic involvement in the disease and broadening our understanding of clinical and molecular implications. This study not only contributes specific insights into LEMD2-related conditions but also underscores potential therapeutic paths for disorders affecting nuclear envelope dynamics.

Identification of tumour regression in neoadjuvantly treated pancreatic cancer is based on divergent and nonspecific criteria.

AIMS: Following the increased use of neoadjuvant therapy for pancreatic cancer, grading of tumour regression (TR) has become part of routine diagnostics. However, it suffers from marked interobserver variation, which is mainly ascribed to the subjectivity of the defining criteria of the categories in TR grading systems. We hypothesized that a further cause for the interobserver variation is the use of divergent and nonspecific morphological criteria to identify tumour regression. METHODS AND RESULTS: Twenty treatment-naïve pancreatic cancers and 20 pancreatic cancers treated with neoadjuvant chemotherapy were reviewed by three experienced pancreatic pathologists who, blinded for treatment status, categorized each tumour as treatment-naïve or neoadjuvantly treated, and annotated all tissue areas they considered showing tumour regression. Only 50%-65% of the cases were categorized correctly, and the annotated tissue areas were highly discrepant (only 3%-41% overlap). When the prevalence of various morphological features deemed to indicate TR was compared between treatment-naïve and neoadjuvantly treated tumours, only one pattern, characterized by reduced cancer cell density and prominent stroma affecting a large area of the tumour bed, occurred significantly more frequently, but not exclusively, in the neoadjuvantly treated group. Finally, stromal features, both morphological and biological, were investigated as possible markers for tumour regression, but failed to distinguish TR from native tumour stroma. CONCLUSION: There is considerable divergence in opinion between pathologists when it comes to the identification of tumour regression. Reliable identification of TR is only possible if it is extensive, while lesser degrees of treatment effect cannot be recognized with certainty.

Ex vivo modeling of acquired drug resistance in BRAF - mutated pancreatic cancer organoids uncovers individual therapeutic vulnerabilities.

Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis due to late detection and limited treatment options. Some PDAC patients harbor alterations that qualify for targeted treatment strategies but develop acquired resistance, leading to treatment failure. We here report the ex vivo modeling of acquired drug resistance by creating a PDAC patient-derived tumor organoid (PDTO) model harboring a rare BRAF R506_K507ins VLR mutation resulting in a resistance to trametinib, a MEK inhibitor. Genomic and transcriptomic analyses revealed upregulated WNT signaling in resistant PDTO clones compared to treatment-naïve parental control cells. By combining genomic and transcriptomic analysis with a functional drug testing approach, we uncovered a de novo upregulation and circumventive reliance on WNT signaling in resistant PDTO clones. Ex vivo models such as PDTOs represent valuable tools for resistance modelling and offer the discovery of novel therapeutic approaches for patients in need where clinical diagnostic tools are currently at the limit.

Limited Value of Bladder Wash Cytology During Follow-Up of Patients With Non-muscle Invasive Bladder Cancer.

Aims We aimed to assess the performance of bladder wash cytology (BWC) in daily clinical practice in a pure follow-up cohort of patients previously diagnosed with non-muscle invasive bladder cancer (NMIBC). Materials and methods We analyzed 2064 BWCs derived from 314 patients followed for NMIBC (2003-2016). Follow-up investigations were performed using cystoscopy (CS) in combination with BWC. Patients with suspicious CS and/or positive BWC underwent bladder biopsy or transurethral resection. BWC was considered positive if malignant or suspicious cells were reported. Sensitivity (Sn) and specificity (Sp) were calculated for the entire cohort and separately for low-grade (LG) and high-grade (HG) tumors, and carcinoma in situ (CIS) subgroups. Results A total of 95 recurrences were detected, of which only three were detected by BWC alone. Overall, Sn and Sp of BWC were 17.9% and 99.5%, respectively. For LG disease, these numbers were 14.0% and 100%, and for HG disease, these were 22.2% and 99.1%, respectively. For patients with CIS at initial diagnosis, Sn and Sp were 11.0% and 71.4%, respectively. For isolated primary CIS, Sn was 50.0%, and Sp was 98.2%. Conclusion Routine use of BWC in the follow-up for NMIBC is of limited value even in HG tumors. In the presence of isolated primary CIS, adjunct BWC might be justified.

Defatting of Human Livers During Long-Term e x situ Normothermic Perfusion: Novel Strategy to Rescue Discarded Organs for Transplantation.

OBJECTIVE: To develop a protocol for the defatting of steatotic liver grafts during long-term ex situ normothermic machine perfusion. BACKGROUND: Despite the alarming increase in donor organ shortage, the highly prevalent fatty liver grafts are often discarded due to the risk of primary nonfunction. Effective strategies preventing such outcomes are currently lacking. An exciting new avenue is the introduction of ex situ normothermic machine perfusion (NMP), enabling a liver to remain fully functional for up to 2 weeks and providing a unique window of opportunity for defatting before transplantation. METHODS: Over a 5-year period, 23 discarded liver grafts and 28 partial livers from our resection program were tested during ex situ normothermic machine perfusion. The steatosis degree was determined on serial biopsies by expert pathologists, and triglyceride contents were measured simultaneously. RESULTS: Of 51 liver grafts, 20 were steatotic, with up to 85% macrovesicular steatosis, and were perfused for up to 12 days. Ten livers displayed marked (5 of which almost complete) loss of fat, while the other 10 did not respond to long-term perfusion. Successful defatting was related to prolonged perfusion, automated glucose control, circadian nutrition, and L-carnitine/fenofibrate supplementation. Pseudopeliotic steatosis and the associated activation of Kupffer/stellate cells were unexpected processes that might contribute to defatting. Synthetic and metabolic functions remained preserved for most grafts until perfusion ended. CONCLUSION: Ex situ long-term perfusion effectively reduces steatosis while preserving organ viability and may in the future allow transplantation of primarily unusable high-risk grafts, significantly increasing the number of organs available for transplantation.

Determinants of Advanced Liver Fibrosis in Adult Patients After Fontan Palliation: Usefulness of Ultrasound Transient Elastography.

BACKGROUND: Fontan-associated liver disease is an increasing concern. Our aim was to assess prevalence and predictors of advanced liver fibrosis with a specific focus on utility of liver stiffness measurement by ultrasound transient elastography. METHODS: A total of 97 adult Fontan patients (55% male, median age: 23.1 years, interquartile range [IQR]: 18.7-30.6); 92 (95%) were evaluated with transient elastography, and 50 (52%) underwent transjugular liver biopsy. Advanced liver fibrosis was defined as congestive hepatic fibrosis score 3 or 4. RESULTS: Only 4 patients (4%) had liver stiffness values < 10 kilopascal (kPa). Liver-stiffness measurements correlated weakly with peak oxygen uptake on exercise testing and Fontan pressure but not with Model for End-Stage Liver Disease excluding INR (MELD-XI) score or spleen size. Serial follow-up liver stiffness measurements in 73 clinically stable patients showed large variability among individual patients. Advanced liver fibrosis was present in 35 of 50 (70%) patients on liver biopsy and was associated to MELD-XI-Score ≥ 11 and splenomegaly but not to liver-stiffness measurements. Advanced liver fibrosis was not associated with patient age or time since Fontan operation but with younger age at completion of Fontan (3.7 years, IQR: 2.3-6.3 vs 6.8 years; IQR: 3.5-12.1; P = 0.037). CONCLUSIONS: In our cohort, advanced liver fibrosis was present in the majority of adult Fontan patients. Liver stiffness as measured by transient elastography was not associated with the degree of liver fibrosis. Because of its high variability on serial measurements, it seems not to be useful for clinical decision making. The unexpected finding that younger age at completion of Fontan was associated with advanced liver fibrosis merits further evaluation.

The Milan system for reporting salivary gland cytopathology-A single-center study of 2156 cases.

BACKGROUND: Fine-needle aspiration cytology (FNAC) represents an important diagnostic tool for the workup of salivary gland (SG) lesions. The Milan System for Reporting Salivary Gland Cytopathology (MSRSGC) is a six-tiered system for standardizing diagnoses and improvement of communication between pathologists and clinicians, providing risk of malignancy (ROM) rates for every category. The aims of the present study were (i) to validate the use of MSRSGC in a large series of SG FNAC in a tertiary center in Switzerland, (ii) to determine ROM for each category and compare them with data from MSRSGC and similar studies, and (iii) to investigate whether there were relevant differences of non-diagnostic results between fine-needle aspirations (FNA) performed by cytopathologists compared to non-cytopathologists. METHODS: The files of the department of Pathology in the University Hospital Zurich (UHZ) were searched for SG FNAC between 2010 and 2019. The MSRSGC guidelines were applied retrospectively. Furthermore, ROM, risk of neoplasia (RON), sensitivity, and specificity were calculated based on the cases with histopathological follow-up. RESULTS: A total of 2156 SG FNAC including 753 cases with histopathological follow-up were evaluated. Generally, ROM was within the range of values provided by MSRSGC, with some minor deviations. Sensitivity was 94.6%, and specificity was 99.3%. CONCLUSIONS: Our study confirms the usefulness of MSRSGC. In addition, it provides a detailed insight into the wide spectrum of SG FNAC. Finally, we showed that the rate of non-diagnostic FNA was significantly lower in FNAs performed by cytopathologists compared to non-cytopathologists.

Utility of BAP1, p16, and Methylthio-Adenosine Phosphorylase Immunohistochemistry in Cytological and Histological Samples of Pleural Mesotheliomas.

INTRODUCTION: In most cases, the diagnostic workup of pleural mesotheliomas (MPMs) starts with cytological examination of pleural effusion, but histology is needed to confirm the diagnosis. The introduction of BAP1 and methylthio-adenosine phosphorylase (MTAP) immunohistochemistry has become a powerful tool to confirm the malignant nature of mesothelial proliferations also in cytological specimens. The objective of this study was to determine the concordance of BAP1, MTAP, and p16 expression between cytological and histological samples of patients with MPM. METHODS: Immunohistochemistry of BAP1, MTAP, and p16 was performed on cytological samples and compared with the corresponding histological specimen of 25 patients with MPM. Inflammatory and stromal cells served as positive internal control for all three markers. In addition, samples of 11 patients with reactive mesothelial proliferations served as an external control group. RESULTS: Loss of BAP1, MTAP, and p16 expression was found in 68%, 72%, and 92% of MPM, respectively. Loss of MTAP was associated with loss of p16 expression in all cases. Concordance of BAP1 between cytological and corresponding histological samples was 100% (kappa coefficient 1; p = 0.008). For MTAP and p16, kappa coefficient was 0.9 (p = 0.01) and 0.8 (p = 0.7788), respectively. CONCLUSIONS: Concordant BAP1, MTAP, and p16 expression is found between cytological and corresponding histological samples, indicating that a reliable diagnosis of MPM can be made on cytology only. Of the three markers, BAP1 and MTAP are most reliable in distinguishing malignant from reactive mesothelial proliferations.

Immunohistochemistry for hepatitis E virus capsid protein cross-reacts with cytomegalovirus-infected cells: a potential diagnostic pitfall.

Immunohistochemistry for hepatitis E virus (HEV) ORF2 (capsid) protein is a powerful tool for tissue-based diagnosis of hepatitis E, particularly useful in evaluating abnormal liver values in immunocompromised patients. We report here a previously unobserved reactivity of the HEV ORF2 antibody to human cytomegalovirus (CMV) proteins and contrast the staining patterns encountered in HEV and CMV infection, respectively. As part of a routine diagnostic work-up, the liver biopsy of an immunocompromised patient with elevated liver values was examined histologically for infection with viruses including CMV and HEV. Cytopathic changes were found, suggestive of CMV infection, which was confirmed by immunohistochemistry. Surprisingly, reactivity of a portion of CMV-infected cells with a mouse monoclonal antibody (clone 1E6) against HEV ORF2 protein was also detected. This observation prompted a screening of 22 further specimens (including liver, gastrointestinal, lung, brain and placental biopsies) with confirmed CMV infection/reactivation. Immunoreactivity of CMV-infected cells with HEV ORF2 antibody was observed in 18 of 23 specimens. While the HEV ORF2 antibody showed cytoplasmic, nuclear and canalicular positivity in hepatitis E cases, positivity in CMV-infected cells was limited to the nucleus. In conclusion, the HEV ORF2 antibody (clone 1E6) shows unexpected immunoreactivity against CMV proteins. In contrast to the hepatitis E staining pattern with cytoplasmic, nuclear and occasional canalicular positivity, reactivity in CMV-infected cells is restricted to the nucleus. Awareness of this cross-reactivity and knowledge of the differences in staining patterns will prevent pathologists from misinterpreting positive HEV ORF2 immunohistochemistry in liver specimens.

Hypoxia and Ezrin Expression in Primary Melanoma Have High Prognostic Relevance.

Hypoxia affects tumor aggressiveness and activates pathways associated with epithelial mesenchymal transition (EMT) which are crucial for tumor progress. In this study, the correlation of hypoxia and EMT with sentinel lymph node status and tumor-specific survival was investigated in primary melanomas. CD34 for capillary count and Hypoxia inducible factor-1α (HIF-1α) as hypoxia indicators as well as Ezrin and L1-Cell Adhesion Molecule (L1CAM), both critical proteins contributing to EMT, were analyzed using immunohistochemistry in 49 melanoma patients with long follow-up (F/U, mean 110 months; range 12−263 months). We found a significant correlation between Breslow tumor thickness and Ezrin expression (p = 0.018). L1CAM expression in primary melanoma was significantly associated with HIF-1α expression (p < 0.0001) and sentinel lymph node metastasis (p = 0.011). Furthermore, low capillary count, reflecting hypoxic condition, was significantly associated with Ezrin expression (p = 0.047) and decreased tumor-specific survival (p = 0.035). In addition, patients with high Ezrin expression in their primary melanoma had a dramatic loss of life early in their F/U period (mean survival time 29 months; range 15−44 month). Our results highlight the relevance of Ezrin, L1CAM and HIF-1α as prognostic markers in melanoma patients. Additionally, we demonstrate that hypoxia in primary melanoma affects EMT and is at least partly responsible for early metastatic dissemination.

SF3B1 facilitates HIF1-signaling and promotes malignancy in pancreatic cancer.

Mutations in the splicing factor SF3B1 are frequently occurring in various cancers and drive tumor progression through the activation of cryptic splice sites in multiple genes. Recent studies also demonstrate a positive correlation between the expression levels of wild-type SF3B1 and tumor malignancy. Here, we demonstrate that SF3B1 is a hypoxia-inducible factor (HIF)-1 target gene that positively regulates HIF1 pathway activity. By physically interacting with HIF1α, SF3B1 facilitates binding of the HIF1 complex to hypoxia response elements (HREs) to activate target gene expression. To further validate the relevance of this mechanism for tumor progression, we show that a reduction in SF3B1 levels via monoallelic deletion of Sf3b1 impedes tumor formation and progression via impaired HIF signaling in a mouse model for pancreatic cancer. Our work uncovers an essential role of SF3B1 in HIF1 signaling, thereby providing a potential explanation for the link between high SF3B1 expression and aggressiveness of solid tumors.

Drug screening and genome editing in human pancreatic cancer organoids identifies drug-gene interactions and candidates for off-label treatment.

Pancreatic cancer (PDAC) is a highly aggressive malignancy for which the identification of novel therapies is urgently needed. Here, we establish a human PDAC organoid biobank from 31 genetically distinct lines, covering a representative range of tumor subtypes, and demonstrate that these reflect the molecular and phenotypic heterogeneity of primary PDAC tissue. We use CRISPR-Cas9 genome editing and drug screening to characterize drug-gene interactions with ARID1A and BRCA2. We find that missense- but not frameshift mutations in the PDAC driver gene ARID1A are associated with increased sensitivity to the kinase inhibitors dasatinib (p < 0.0001) and VE-821 (p < 0.0001). We conduct an automated drug-repurposing screen with 1,172 FDA-approved compounds, identifying 26 compounds that effectively kill PDAC organoids, including 19 chemotherapy drugs currently approved for other cancer types. We validate the activity of these compounds in vitro and in vivo. The in vivo validated hits include emetine and ouabain, compounds which are approved for non-cancer indications and which perturb the ability of PDAC organoids to respond to hypoxia. Our study provides proof-of-concept for advancing precision oncology and identifying candidates for drug repurposing via genome editing and drug screening in tumor organoid biobanks.

De novo expression of gastrokines in pancreatic precursor lesions impede the development of pancreatic cancer.

Molecular events occurring in stepwise progression from pre-malignant lesions (pancreatic intraepithelial neoplasia; PanIN) to the development of pancreatic ductal adenocarcinoma (PDAC) are poorly understood. Thus, characterization of early PanIN lesions may reveal markers that can help in diagnosing PDAC at an early stage and allow understanding the pathology of the disease. We performed the molecular and histological assessment of patient-derived PanINs, tumor tissues and pancreas from mouse models with PDAC (KC mice that harbor K-RAS mutation in pancreatic tissue), where we noted marked upregulation of gastrokine (GKN) proteins. To further understand the role of gastrokine proteins in PDAC development, GKN-deficient KC mice were developed by intercrossing gastrokine-deficient mice with KC mice. Panc-02 (pancreatic cancer cells of mouse origin) were genetically modified to express GKN1 for further in vitro and in vivo analysis. Our results show that gastrokine proteins were absent in healthy pancreas and invasive cancer, while its expression was prominent in low-grade PanINs. We could detect these proteins in pancreatic juice and serum of KC mice. Furthermore, accelerated PanIN and tumor development were noted in gastrokine deficient KC mice. Loss of gastrokine 1 protein delayed apoptosis during carcinogenesis leading to the development of desmoplastic stroma while loss of gastrokine 2 increased the proliferation rate in precursor lesions. In summary, we identified gastrokine proteins in early pancreatic precursor lesions, where gastrokine proteins delay pancreatic carcinogenesis.

ß6-Integrin Serves as a Potential Serum Marker for Diagnosis and Prognosis of Pancreatic Adenocarcinoma.

INTRODUCTION: Despite enormous efforts during the past decades, pancreatic adenocarcinoma (PAC) remains one of the most deleterious cancer entities. A useful biomarker for early detection or prognosis of PAC does not yet exist. The goal of our study was the characterization of ß6-integrin (ITGB6) as a novel serum tumor marker for refined diagnosis and prognosis of PAC. Serum ITGB6 levels were analyzed in 3 independent PAC cohorts consisting of retrospectively and prospectively collected serum and/or (metastatic) PAC tissue specimens. METHODS: Using 2 independent cohorts, we measured serum ITGB6 concentrations in 10 chronic pancreatitis patients, 10 controls, as well as in 27 (cohort 1) and 24 (cohort 2) patients with PAC, respectively. In these patients, we investigated whether ITGB6 serum levels correlate with known clinical and prognostic markers for PAC and whether they might differ between patients with PAC or benign inflammatory diseases of the pancreas. RESULTS: We found that elevated serum ITGB6 levels (≥0.100 ng/mL) in patients suffering from metastasizing PAC presented an unfavorable prognostic outcome. By correlating the ITGB6 tissue expression in primary and metastatic PAC with clinical parameters, we found that positive ITGB6 expression in the tumor tissue is linked to increased serum ITGB6 levels in nonmetastatic PAC and correlates with carbohydrate antigen 19-9 and clinical outcome. DISCUSSION: Our findings suggest that ITGB6 might serve as a novel serum biomarker for early diagnosis and prognosis of PAC. Given the limited specificity and sensitivity of currently used carbohydrate antigen 19-9-based assays, ITGB6 may have the potential to improve the diagnostic accuracy for PAC.

In vivo adenine base editing of PCSK9 in macaques reduces LDL cholesterol levels.

Most known pathogenic point mutations in humans are C•G to T•A substitutions, which can be directly repaired by adenine base editors (ABEs). In this study, we investigated the efficacy and safety of ABEs in the livers of mice and cynomolgus macaques for the reduction of blood low-density lipoprotein (LDL) levels. Lipid nanoparticle-based delivery of mRNA encoding an ABE and a single-guide RNA targeting PCSK9, a negative regulator of LDL, induced up to 67% editing (on average, 61%) in mice and up to 34% editing (on average, 26%) in macaques. Plasma PCSK9 and LDL levels were stably reduced by 95% and 58% in mice and by 32% and 14% in macaques, respectively. ABE mRNA was cleared rapidly, and no off-target mutations in genomic DNA were found. Re-dosing in macaques did not increase editing, possibly owing to the detected humoral immune response to ABE upon treatment. These findings support further investigation of ABEs to treat patients with monogenic liver diseases.

Secondary sclerosing cholangitis as cause of persistent jaundice in patients with severe COVID-19.

BACKGROUND & AIMS: Little is known about cholestasis, including its most severe variant secondary sclerosing cholangitis (SSC), in critically ill patients with coronavirus disease 19 (COVID-19). In this study, we analysed the occurrence of cholestatic liver injury and SSC, including clinical, serological, radiological and histopathological findings. METHODS: We conducted a retrospective single-centre analysis of all consecutive patients admitted to the intensive care unit (ICU) as a result of severe COVID-19 at the University Hospital Zurich to describe cholestatic injury in these patients. The findings were compared to a retrospective cohort of patients with severe influenza A. RESULTS: A total of 34 patients with severe COVID-19 admitted to the ICU were included. Of these, 14 patients (41%) had no cholestasis (group 0), 11 patients (32%, group 1) developed mild and 9 patients (27%, group 2) severe cholestasis. Patients in group 2 had a more complicated disease course indicated by significantly longer ICU stay (median 51 days, IQR 25-86.5) than the other groups (group 0: median 9.5 days, IQR 3.8-18.3, P = .001; and group 1: median 16 days, IQR 8-30, P < .05 respectively). Four patients in group 2 developed SSC compared to none in the influenza A cohort. The available histopathological findings suggest an ischaemic damage to the perihilar bile ducts. CONCLUSIONS: The development of SSC represents an important complication of critically ill COVID-19 patients and needs to be considered in the diagnostic work up in prolonged cholestasis. The occurrence of SSC is of interest in the ongoing pandemic since it is associated with considerable morbidity and mortality.

Autochthonous hepatitis E as a cause of acute-on-chronic liver failure and death: histopathology can be misleading but transaminases may provide a clue.

BACKGROUND AND AIM: Acute decompensation and death have been observed in patients with acute hepatitis E virus (HEV) infection and preexisting liver cirrhosis. However, the clinical, laboratory and histological features need to be fully characterised. METHODS: Some of us recently described the histological presentation of hepatitis E in a large panel of liver tissue specimens. Here, we conducted a case-control study to investigate the clinical and laboratory features of the subset of patients with HEV-related acute-on-chronic liver failure (ACLF) and death. Each patient was matched to three control patients with histologically confirmed severe alcoholic hepatitis based on sex, age, total bilirubin, INR, serum creatinine and MELD score on admission. RESULTS: Of 5 patients who died in a context of HEV-related ACLF, 3 (60%) were male and the median age was 66 years (range 51–76). Median alanine aminotransferase (ALT) at presentation was 2610 U/l (range 705–3134) and aspartate aminotransferase (AST) 2818 U/l (range 1176–8611). Liver function was heavily altered in all patients. Histological analyses revealed steatohepatitis on a background of cirrhosis, suggestive of an alcoholic or nonalcoholic origin. Based on histopathology, alcoholic hepatitis was initially suspected in two patients and corticosteroid treatment was initiated. Ribavirin was started in four patients. Median time from hospitalisation to death was 17 days (range 6–25 days). AST levels in patients with HEV-related ACLF were significantly higher as compared to the matched patients with severe alcoholic hepatitis. CONCLUSION: Typical histopathological features of viral hepatitis may be absent in ACLF caused by HEV infection. HEV infection should be sought in acute decompensation of cirrhosis and ACLF even in the absence of histological changes suggesting viral infection.

NASH limits anti-tumour surveillance in immunotherapy-treated HCC.

Hepatocellular carcinoma (HCC) can have viral or non-viral causes1-5. Non-alcoholic steatohepatitis (NASH) is an important driver of HCC. Immunotherapy has been approved for treating HCC, but biomarker-based stratification of patients for optimal response to therapy is an unmet need6,7. Here we report the progressive accumulation of exhausted, unconventionally activated CD8+PD1+ T cells in NASH-affected livers. In preclinical models of NASH-induced HCC, therapeutic immunotherapy targeted at programmed death-1 (PD1) expanded activated CD8+PD1+ T cells within tumours but did not lead to tumour regression, which indicates that tumour immune surveillance was impaired. When given prophylactically, anti-PD1 treatment led to an increase in the incidence of NASH-HCC and in the number and size of tumour nodules, which correlated with increased hepatic CD8+PD1+CXCR6+, TOX+, and TNF+ T cells. The increase in HCC triggered by anti-PD1 treatment was prevented by depletion of CD8+ T cells or TNF neutralization, suggesting that CD8+ T cells help to induce NASH-HCC, rather than invigorating or executing immune surveillance. We found similar phenotypic and functional profiles in hepatic CD8+PD1+ T cells from humans with NAFLD or NASH. A meta-analysis of three randomized phase III clinical trials that tested inhibitors of PDL1 (programmed death-ligand 1) or PD1 in more than 1,600 patients with advanced HCC revealed that immune therapy did not improve survival in patients with non-viral HCC. In two additional cohorts, patients with NASH-driven HCC who received anti-PD1 or anti-PDL1 treatment showed reduced overall survival compared to patients with other aetiologies. Collectively, these data show that non-viral HCC, and particularly NASH-HCC, might be less responsive to immunotherapy, probably owing to NASH-related aberrant T cell activation causing tissue damage that leads to impaired immune surveillance. Our data provide a rationale for stratification of patients with HCC according to underlying aetiology in studies of immunotherapy as a primary or adjuvant treatment.

Morphological Heterogeneity in Pancreatic Cancer Reflects Structural and Functional Divergence.

Inter- and intratumor heterogeneity is an important cause of treatment failure. In human pancreatic cancer (PC), heterogeneity has been investigated almost exclusively at the genomic and transcriptional level. Morphological heterogeneity, though prominent and potentially easily assessable in clinical practice, remains unexplored. This proof-of-concept study aims at demonstrating that morphological heterogeneity reflects structural and functional divergence. From the wide morphological spectrum of conventional PC, four common and distinctive patterns were investigated in 233 foci from 39 surgical specimens. Twenty-six features involved in key biological processes in PC were analyzed (immuno-)histochemically and morphometrically: cancer cell proliferation (Ki67) and migration (collagen fiber alignment, MMP14), cancer stem cells (CD44, CD133, ALDH1), amount, composition and spatial arrangement of extracellular matrix (epithelial proximity, total collagen, collagen I and III, fibronectin, hyaluronan), cancer-associated fibroblasts (density, αSMA), and cancer-stroma interactions (integrins α2, α5, α1; caveolin-1). All features differed significantly between at least two of the patterns. Stromal and cancer-cell-related features co-varied with morphology and allowed prediction of the morphological pattern. In conclusion, morphological heterogeneity in the cancer-cell and stromal compartments of PC correlates with structural and functional diversity. As such, histopathology has the potential to inform on the operationality of key biological processes in individual tumors.

Pancreatic fistulas following distal pancreatectomy are unrelated to the texture quality of the pancreas.

PURPOSE: The relevance of pancreatic texture for pancreatic fistula (POPF) formation after distal pancreatectomy (DP) remains ill defined. Recent POPF definition adjustments and common subjective pancreatic texture assessment are further drawbacks in the investigation of pancreatic texture as a factor for POPF development after DP. METHODS: The predictive value of pancreatic texture by histologic assessment was investigated for POPF formation after DP, respecting the updated 2016 fistula definition. Histologic evaluation at the resection margin included amount of steatosis, degree of fibrosis, and pancreatic duct size. RESULTS: A total of 102 patients who underwent DP were included. Thirty-six patients developed POPF. There was no difference in histologic variables in patients with and without POPF. In the univariate analysis, none of the three histologic features showed significant correlation with POPF formation. The ROC (receiver operating characteristic) curve demonstrated poor utility for the grade of steatosis 0.481 ± 0.058 (p = 0.75) and grade of fibrosis 0.466 ± 0.058 (p = 0.57) as predictive factors for POPF formation. CONCLUSION: Results indicate that pancreatic texture does not predict POPF formation following DP. This is particularly relevant in the context of the increasing use of robotic and laparoscopic approaches for DPs with limited clinical pancreatic texture assessment by palpation.