Small duct and large duct type intrahepatic cholangiocarcinoma reveal distinct patterns of immune signatures.

PURPOSE: Dedicated gene signatures in small (SD-iCCA) and large (LD-iCCA) duct type intrahepatic cholangiocarcinoma remain unknown. We performed immune profiling in SD- and LD-iCCA to identify novel biomarker candidates for personalized medicine. METHODS: Retrospectively, 19 iCCA patients with either SD-iCCA (n = 10, median age, 63.1 years (45-86); men, 4) or LD-iCCA (n = 9, median age, 69.7 years (62-85); men, 5)) were included. All patients were diagnosed and histologically confirmed between 04/2009 and 01/2021. Tumor tissue samples were processed for differential expression profiling using NanoString nCounter® PanCancer Immune Profiling Panel. RESULTS: With the exception of complement signatures, immune-related pathways were broadly downregulated in SD-iCCA vs. LD-iCCA. A total of 20 immune-related genes were strongly downregulated in SD-iCCA with DMBT1 (log2fc = -5.39, p = 0.01) and CEACAM6 (log2fc = -6.38, p = 0.01) showing the strongest downregulation. Among 7 strongly (log2fc > 2, p ≤ 0.02) upregulated genes, CRP (log2fc = 5.06, p = 0.02) ranked first, and four others were associated with complement (C5, C4BPA, C8A, C8B). Total tumor-infiltrating lymphocytes (TIL) signature was decreased in SD-iCCA with elevated ratios of exhausted-CD8/TILs, NK/TILs, and cytotoxic cells/TILs while having decreased ratios of B-cells/TILs, mast cells/TILs and dendritic cells/TILs. The immune profiling signatures in SD-iCCA revealed downregulation in chemokine signaling pathways inclulding JAK2/3 and ERK1/2 as well as nearly all cytokine-cytokine receptor interaction pathways with the exception of the CXCL1/CXCR1-axis. CONCLUSION: Immune patterns differed in SD-iCCA versus LD-iCCA. We identified potential biomarker candidate genes, including CRP, CEACAM6, DMBT1, and various complement factors that could be explored for augmented diagnostics and treatment decision-making.

Impaired HBsAg release and antiproliferative/antioxidant cell regulation by HBeAg-negative patient isolates reflects an evolutionary process.

BACKGROUND: The hepatitis B e antigen (HBeAg)-negative infection Phase 3 is characterized by no or minimal signs of hepatic inflammation and the absence of hepatic fibrosis. However, underlying molecular mechanisms leading to this benign phenotype are poorly understood. METHODS: Genotype A, B and D HBeAg-negative patient isolates with precore mutation G1896A from Phase 3 were analysed in comparison with respective HBeAg-positive rescue mutant and HBeAg-positive wild-type reference genomes regarding differences in viral replication, morphogenesis, infectivity and impact on NF-E2-related factor 2 (Nrf2)/antioxidant response element (ARE)-dependent gene expression and cellular kinome. RESULTS: In comparison with reference genomes, the patient isolates are characterized by a lower intra- and extracellular hepatitis B surface antigen (HBsAg)-amount, and HBsAg-retention in the endoplasmic reticulum. Rescue of HBeAg expression increased HBsAg-amount but not its release. Expression of the isolated genomes is associated with a higher Nrf2/ARE-dependent gene expression as compared to reference genomes independent of HBeAg expression. Kinome analyses revealed a decreased activity of receptors involved in regulation of proliferative pathways for all patient isolates compared to the reference genomes. No specific conserved mutations could be found between all genomes from Phase 3. CONCLUSIONS: HBeAg-negative genomes from Phase 3 exhibit distinct molecular characteristics leading to lower HBsAg synthesis and release, enhanced oxidative stress protection and decreased activity of key kinases, triggering an antiproliferative stage, which might contribute to the lower probability of hepatocellular carcinoma. The observed differences cannot be associated with loss of HBeAg or specific mutations common to all analysed isolates, indicating the phenotype of Phase 3 derived genomes to be the result of a multifactorial process likely reflecting a conserved natural selection process.

Molecular insights into the regulatory landscape of PKC-related kinase-2 (PRK2/PKN2) using targeted small compounds.

The PKC-related kinases (PRKs, also termed PKNs) are important in cell migration, cancer, hepatitis C infection, and nutrient sensing. They belong to a group of protein kinases called AGC kinases that share common features like a C-terminal extension to the catalytic domain comprising a hydrophobic motif. PRKs are regulated by N-terminal domains, a pseudosubstrate sequence, Rho-binding domains, and a C2 domain involved in inhibition and dimerization, while Rho and lipids are activators. We investigated the allosteric regulation of PRK2 and its interaction with its upstream kinase PDK1 using a chemical biology approach. We confirmed the phosphoinositide-dependent protein kinase 1 (PDK1)-interacting fragment (PIF)-mediated docking interaction of PRK2 with PDK1 and showed that this interaction can be modulated allosterically. We showed that the polypeptide PIFtide and a small compound binding to the PIF-pocket of PRK2 were allosteric activators, by displacing the pseudosubstrate PKL region from the active site. In addition, a small compound binding to the PIF-pocket allosterically inhibited the catalytic activity of PRK2. Together, we confirmed the docking interaction and allostery between PRK2 and PDK1 and described an allosteric communication between the PIF-pocket and the active site of PRK2, both modulating the conformation of the ATP-binding site and the pseudosubstrate PKL-binding site. Our study highlights the allosteric modulation of the activity and the conformation of PRK2 in addition to the existence of at least two different complexes between PRK2 and its upstream kinase PDK1. Finally, the study highlights the potential for developing allosteric drugs to modulate PRK2 kinase conformations and catalytic activity.

Functional analysis of MMR gene VUS from potential Lynch syndrome patients.

Lynch syndrome is caused by inactivating variants in DNA mismatch repair genes, namely MLH1, MSH2, MSH6 and PMS2. We have investigated five MLH1 and one MSH2 variants that we have identified in Turkish and Tunisian colorectal cancer patients. These variants comprised two small deletions causing frameshifts resulting in premature stops which could be classified pathogenic (MLH1 p.(His727Profs*57) and MSH2 p.(Thr788Asnfs*11)), but also two missense variants (MLH1 p.(Asn338Ser) and p.(Gly181Ser)) and two small, in-frame deletion variants (p.(Val647-Leu650del) and p.(Lys678_Cys680del)). For such small coding genetic variants, it is unclear if they are inactivating or not. We here provide clinical description of the variant carriers and their families, and we performed biochemical laboratory testing on the variant proteins to test if their stability or their MMR activity are compromised. Subsequently, we compared the results to in-silico predictions on structure and conservation. We demonstrate that neither missense alteration affected function, while both deletion variants caused a dramatic instability of the MLH1 protein, resulting in MMR deficiency. These results were consistent with the structural analyses that were performed. The study shows that knowledge of protein function may provide molecular explanations of results obtained with functional biochemical testing and can thereby, in conjunction with clinical information, elevate the evidential value and facilitate clinical management in affected families.

Tumour-specific activation of a tumour-blood transport improves the diagnostic accuracy of blood tumour markers in mice.

BACKGROUND: The accuracy of blood-based early tumour recognition is compromised by signal production at non-tumoral sites, low amount of signal produced by small tumours, and variable tumour production. Here we examined whether tumour-specific enhancement of vascular permeability by the particular tumour homing peptide, iRGD, which carries dual function of binding to integrin receptors overexpressed in the tumour vasculature and is known to promote extravasation via neuropilin-1 receptor upon site-specific cleavage, might be useful to improve blood-based tumour detection by inducing a yet unrecognised vice versa tumour-to-blood transport. METHODS: To detect an iRGD-induced tumour-to-blood transport, we examined the effect of intravenously injected iRGD on blood levels of α-fetoprotein (AFP) and autotaxin in several mouse models of hepatocellular carcinoma (HCC) or in mice with chronic liver injury without HCC, and on prostate-specific antigen (PSA) levels in mice with prostate cancer. FINDINGS: Intravenously injected iRGD rapidly and robustly elevated the blood levels of AFP in several mouse models of HCC, but not in mice with chronic liver injury. The effect was primarily seen in mice with small tumours and normal basal blood AFP levels, was attenuated by an anti-neuropilin-1 antibody, and depended on the concentration gradient between tumour and blood. iRGD treatment was also able to increase blood levels of autotaxin in HCC mice, and of PSA in mice with prostate cancer. INTERPRETATION: We conclude that iRGD induces a tumour-to-blood transport in a tumour-specific fashion that has potential of improving diagnosis of early stage cancer. FUNDING: Deutsche Krebshilfe, DKTK, LOEWE-Frankfurt Cancer Institute.

Erk Inhibition as a Promising Therapeutic Strategy for High IL-8-Secreting and Low SPTAN1-Expressing Colorectal Cancer.

Tumor recurrence and drug resistance are responsible for poor prognosis in colorectal cancer (CRC). DNA mismatch repair (MMR) deficiency or elevated interleukin-8 (IL-8) levels are characteristics of CRCs, which have been independently correlated with treatment resistance to common therapies. We recently demonstrated significantly impaired therapeutical response and increased IL-8 release of CRC cell lines with reduced expression of MMR protein MLH1 as well as cytoskeletal non-erythrocytic spectrin alpha II (SPTAN1). In the present study, decreased intratumoral MLH1 and SPTAN1 expression in CRCs could be significantly correlated with enhanced serum IL-8. Furthermore, using stably reduced SPTAN1-expressing SW480, SW620 or HT-29 cell lines, the RAS-mediated RAF/MEK/ERK pathway was analyzed. Here, a close connection between low SPTAN1 expression, increased IL-8 secretion, enhanced extracellular-signal-regulated kinase (ERK) phosphorylation and a mesenchymal phenotype were detected. The inhibition of ERK by U0126 led to a significant reduction in IL-8 secretion, and the combination therapy of U0126 with FOLFOX optimizes the response of corresponding cancer cell lines. Therefore, we hypothesize that the combination therapy of FOLFOX and U0126 may have great potential to improve drug efficacy on this subgroup of CRCs, showing decreased MLH1 and SPTAN1 accompanied with high serum IL-8 in affected patients.

Interventional Treatments of Colorectal Liver Metastases Using Thermal Ablation and Transarterial Chemoembolization: A Single-Center Experience over 26 Years.

The aim of this study was to analyze the long-term results of different locoregional treatments for colorectal cancer liver metastases (CRLM), including transarterial chemoembolization (TACE), laser-induced thermotherapy (LITT) and microwave ablation (MWA). A total of 2140 patients with CRLM treated at our department between 1993 and 2020 were included in this retrospective study. The patients were divided into the following groups: LITT (573 patients; median age: 62 years), TACE + LITT (346 patients; median age: 62 years), MWA (67 patients; median age: 59 years), TACE + MWA (152 patients; median age: 65 years), and TACE (1002 patients; median age: 62 years). Median survival was 1.9 years in the LITT group and 1.7 years in the TACE + LITT group. The median survival times in the MWA group and TACE + MWA group were 3.1 years and 2.1 years, respectively. The median survival in the TACE group was 0.8 years. The 1-, 3-, and 5-year survival rates were 77%, 27%, and 9% in the LITT group and 74%, 18%, and 5% in the TACE + LITT group, respectively. The corresponding survival rates were 80%, 55%, and 33% in the MWA group, 74%, 36%, and 20% in the TACE + MWA group and 37%, 3%, and 0% in the TACE group, respectively. The long-term results of this study demonstrate the efficacy of locoregional treatments in treating patients with CRLM. The longest survival was found in the MWA group, followed by the combination therapy of TACE and MWA.

Diagnostic performance of non-invasive liver fibrosis scores in patients with early-intermediate hepatocellular carcinoma.

PURPOSE: Hepatocellular carcinoma (HCC) arises in individuals with underlying liver disease. Diagnosing the degree of hepatic fibrosis helps to determine the severity of the underlying liver disease and may influence therapeutic decisions in HCC patients. Non-invasive fibrosis scores can be used to estimate the degree of fibrosis in liver disease patients, but most of these scores were developed in patients with viral hepatitis and without HCC. This study explored the ability of the Fibrosis-4 Index (FIB-4), the AST/Platelet Ratio Index (APRI), and the AST/ALT ratio to diagnose or exclude advanced fibrosis (METAVIR F3/4 versus F0-2) in patients with early-intermediate, potentially resectable HCC. METHODS: We retrospectively reviewed 119 patients who underwent hepatic resection for HCC at a tertiary centre (2007-2019), 75 of whom had advanced fibrosis (prevalence 63%). Histological assessment of the surgical liver specimen was used as a reference standard for the degree of fibrosis. RESULTS: Overall diagnostic performance was highest for the FIB-4 Index, with an area under the receiver operating characteristic curve (AUROC) of 0.82, compared with 0.78 for APRI, and 0.56 for the AST/ALT ratio. Using established cut-off values, FIB-4 achieved a 90% positive predictive value at the higher cut-off (3.25) and a 90% negative predictive value at the lower cut-off (1.45). CONCLUSION: The FIB-4 Index could reliably diagnose or exclude advanced fibrosis in patients with early-intermediate HCC, and may thus have a role in guiding therapeutic decisions in these patients.

[Iron supplementation in patients with chronic inflammatory bowel disease: recommendations for a practical approach]. / Eisensupplementation bei Patienten mit chronisch-entzündlicher Darmerkrankung: Empfehlungen zur praxisnahen Vorgehensweise.

Iron deficiency is the predominant cause of anemia. Iron deficiency anemia plays a major role, especially in patients with inflammatory bowel disease (IBD), and is the most common extraintestinal manifestation and IBD-associated systemic complication. The presence of anemia leads to a reduction in quality of life in patients with IBD associated with limitations in physical, emotional, and cognitive function. In addition, it is associated with an increased hospitalization rate. For this reason, iron supplementation is of particular importance. Oral and intravenous iron supplements are used to treat iron deficiency. Due to the lack of absorption and gastrointestinal side effects of oral substitution, intravenous supplementation is becoming increasingly important. However, there are still certain concerns about intravenous administration.With the help of this review, we want to address the topic of iron substitution in patients with IBD, summarize current guideline recommendations, and provide a practical approach.

Real-world effectiveness of voxilaprevir/velpatasvir/sofosbuvir in patients following DAA failure.

Background & Aims: Voxilaprevir/velpatasvir/sofosbuvir (VOX/VEL/SOF) is highly effective for re-treatment of direct-acting antiviral (DAA)-experienced patients with chronic HCV infection. In the present study, predictors of virologic treatment response were analyzed in an integrative analysis of three large real-world cohorts. Methods: Consecutive patients re-treated with VOX/VEL/SOF after DAA failure were enrolled between 2016 and 2021 in Austria, Belgium, Germany, Italy, Spain and Switzerland. Results: A total of 746 patients were included: median age was 56 (16-88) years and 77% were male. Most patients were infected with HCV genotype 1 (56%) and 3 (32%). 86% of patients carried resistance-associated substitutions in the NS3, NS5A or NS5B regions. Overall, 95.4% (683/716) of patients achieved a sustained virologic response. Treatment effectiveness was significantly affected by advanced liver disease (p <0.001), hepatocellular carcinoma (p <0.001), higher baseline ALT levels (p = 0.02), HCV genotype 3 (p <0.001), and prior VEL/SOF treatment (p = 0.01). In a multivariate analysis, only HCV genotype 3, hepatocellular carcinoma and cirrhosis turned out to be independent predictors of treatment failure. Resistance-associated substitutions, as well as the presence of rare genotypes, did not impact treatment outcome. The effectiveness of rescue therapy with glecaprevir/pibrentasvir and SOF, with or without ribavirin, for 12 to 24 weeks was found to be high (100%). Conclusions: Infection with HCV genotype 3, the presence of liver cancer and cirrhosis are independently associated with failure of VOX/VEL/SOF re-treatment. It is unclear whether the addition of ribavirin and/or extension of treatment duration may be effective to avoid virologic relapse on VOX/VEL/SOF. However, rescue treatment with glecaprevir/pibrentasvir+SOF seems to be effective. Impact and implications: Representative data on the effectiveness of voxilaprevir/velpatasvir/sofosbuvir (VOX/VEL/SOF) in clinical practice are still scarce and the collection of a larger number of patients with difficult-to-treat cofactors including the assessment of resistance-associated substitution profiles is required before more specific recommendations for optimal re-treatment in these patients can be given. Thus, we aimed to analyze treatment effectiveness and predictors of virologic response to VOX/VEL/SOF in an integrative analysis of three large real-word cohorts. The study results, derived from a multicenter cohort consisting of 746 patients, demonstrated that re-treatment with VOX/VEL/SOF is an effective salvage therapy associated with an overall per protocol sustained virologic response rate of 95%. Hepatocellular carcinoma onset, cirrhosis and HCV genotype 3 were identified as independent negative predictors of treatment response, whereas resistance-associated substitutions, as well as rare genotypes and chimera, did not impact sustained virologic response rates following re-treatment with VOX/VEL/SOF.

Heterogeneity of small duct- and large duct-type intrahepatic cholangiocarcinoma.

AIMS: The histological subtype of intrahepatic cholangiocarcinoma (iCCA) is associated with different mutational characteristics that impact clinical management. So far, data are lacking on the presence of small duct iCCA (SD-iCCA) and large duct iCCA (LD-iCCA) in a single patient. The aim of the current study was to determine the presence and degree of intratumoural heterogeneity of SD- and LD-iCCA features in different tumour regions. METHODS AND RESULTS: All patients treated with surgically resected iCCA at Frankfurt University Hospital between December 2005 and March 2023 were retrospectively analysed. Histomorphological features of SD- and LD-iCCA were evaluated by an expert hepatobiliary pathologist. Tissue samples suspicious for subtype heterogeneity were further investigated. Immunohistochemistry for N-cadherin, S100P, MUC5AC, MUC6, TFF1 and AGR2 and mutational profiling with the Illumina TruSight Oncology 500 (TSO500) assay were performed separately for the SD- and LD-iCCA regions. Of 129 patients with surgically resected iCCA, features of either SD- or LD-iCCA were present in 67.4% (n = 87) and 24.8% of the patients (n = 32), respectively; 7.8% (n = 10) had histomorphological features of both SD- and LD-iCCA, seven patients (5.4%) of which had sufficient formalin-fixed, paraffin-embedded tissue for further analysis. Heterogeneity of both subtypes could be confirmed with immunohistochemistry. In five of seven (71.4%) patients, molecular profiling revealed intratumoural differences in genetic alterations between the SD- and LD-iCCA region. In one patient, a BRAF mutation (p.V600E) was found in the SD-iCCA but not in the LD-iCCA region of the tumour. CONCLUSIONS: A marked portion of patients with iCCA exhibits both SD- and LD-iCCA in different tumour regions. In case of the presence of histopathological heterogeneity, mutational profiling should be considered to avoid missing therapeutically relevant genetic alterations.

Is a higher frequency of esophageal dilations more effective in treating benign esophageal strictures? Retrospective, multicenter study.

Background and study aims There is still a lack of evidence-based recommendations concerning endoscopic bougienage in benign esophageal strictures. Our study aimed to assess the relevance of the time interval between endoscopic dilation (ED) sessions with regard to endoscopic and clinical response. Patients and methods We performed a retrospective study including patients treated with endoscopic bougienage for a benign esophageal stricture in two German centers. Primary endpoint was the number of ED until freedom from dysphagia was achieved. Secondary endpoints were analyses on reaching a diameter of 15 mm and on achieving clinical freedom from symptoms. Results Between April 2014 and March 2020, bougienage was used as the primary treatment for benign esophageal strictures in 238 patients (194 patients in Center 1; 44 patients in Center 2). Both centers differed in their endoscopic bougienage regime: Center 1 was characterized by a higher frequency of interventions compared to Center 2 (median: 2 days [range 1-28] vs. 10 days [range 1-41]; P <0.001). Clinical response was achieved significantly earlier using the high-frequency regimen in all patients except for those with post-radiogen strictures, who clinically benefited from a low-frequency ED program. Accordingly, patients receiving higher-frequency ED reached a significantly larger post-dilation diameter and considerably larger diameter differences. Conclusions The results of our study demonstrate that a treatment concept consisting of higher-frequency bougienages seems to be more effective in treating most types of esophageal stricture. Radiogenic strictures were the only types of stenoses that benefited from a lower frequency ED program.

Five-year follow-up of 96 weeks peginterferon plus tenofovir disoproxil fumarate in hepatitis D.

BACKGROUND & AIMS: Until recently, pegylated interferon-alfa-2a (PEG-IFNa) therapy was the only treatment option for patients infected with hepatitis D virus (HDV). Treatment with PEG-IFNa with or without tenofovir disoproxil fumarate (TDF) for 96 weeks resulted in HDV RNA suppression in 44% of patients at the end of therapy but did not prevent short-term relapses within 24 weeks. The virological and clinical long-term effects after prolonged PEG-IFNa-based treatment of hepatitis D are unknown. METHODS: In the HIDIT-II study patients (including 40% with liver cirrhosis) received 180 µg PEG-IFNa weekly plus 300 mg TDF once daily (n = 59) or 180 µg PEG-IFNa weekly plus placebo (n = 61) for 96 weeks. Patients were followed until week 356 (5 years after end of therapy). RESULTS: Until the end of follow-up, 16 (13%) patients developed liver-related complications (PEG-IFNa + TDF, n = 5 vs PEG-IFNa + placebo, n = 11; p = .179). Achieving HDV suppression at week 96 was associated with decreased long-term risk for the development of hepatocellular carcinoma (p = .04) and hepatic decompensation (p = .009). Including complications irrespective of PEG-IFNa retreatment status, the number of patients developing serious complications was similar with (3/18) and without retreatment with PEG-IFNa (16/102, p > .999) but was associated with a higher chance of HDV-RNA suppression (p = .024, odds ratio 3.9 [1.3-12]). CONCLUSIONS: Liver-related clinical events were infrequent and occurred less frequently in patients with virological responses to PEG-IFNa treatment. PEG-IFNa treatment should be recommended to HDV-infected patients until alternative therapies become available. Retreatment with PEG-IFNa should be considered for patients with inadequate response to the first course of treatment. CLINICAL TRIAL REGISTRATION: NCT00932971.

Adeno-associated viruses for gene therapy - clinical implications and liver-related complications, a guide for hepatologists.

Gene therapy has garnered increasing interest over recent decades. Several therapies employing gene transfer mechanisms have been developed, and, of these, adeno-associated virus (AAV) vectors have demonstrated viability for use with in vivo gene therapy. Several AAV-based therapeutics have received regulatory approval in the last few years including those for retinal disease, spinal muscular atrophy or aromatic L-amino acid decarboxylase deficiency. Lately, with the introduction of novel liver-directed AAV vector-based therapeutics for the treatment of haemophilia A and B, gene therapy has attracted significant attention in the hepatology community, with the liver increasingly recognised as a target for gene therapy. However, the introduction of foreign DNA into hepatocytes is associated with a risk of hepatic reactions, with raised ALT (alanine aminotransferase) and AST (aspartate aminotransferase) being - so far - the most commonly reported side effects. The complete mechanisms underlying the ALT flairs remain to be determined and the long-term risks associated with these new treatments is not yet known. The liver community is increasingly being asked to support liver-directed gene therapy to mitigate potential liver associated harm. In this review, we focus on AAV vector-based gene therapy, shedding light on this promising technique and its remarkable success in haemophilia, with a special focus on hepatic complications and their management in daily clinical practice.

Short Versus Long Antibiotic Therapy and Risk of Recurrence of Acute Cholangitis Due to Malignant Biliary Strictures.

Malignancies can cause severe stenosis of the biliary tract and therefore predispose a patient to bacterial cholangitis. Upon endoscopic drainage, antibiotic therapy (AT) is performed according to individual clinical judgement, as the optimal duration of AT is unclear to date, especially in the case of multidrug-resistant organisms (MDROs). In a case-based retrospective study, patients with malignant biliary strictures and acute cholangitis were included upon endoscopic retrograde cholangiography (ERC). The outcome of cases treated with short AT (≤6 days) was compared to that of long AT (≥7 days). Recurrent cholangitis (RC) before scheduled stent exchange was the primary end point. In total, 124 patients were included, with 183 cases of proven cholangitis in total. The overall median duration of AT was 7 days (range 1-20), with 74 cases (40%) receiving short AT and 109 (60%) receiving long AT. Short AT was not an independent risk factor for RC (HR = 0.66, p > 0.2), while colonization with MDROs was associated with a higher risk of RC (HR = 2.21, p = 0.005). Placement of a metal stent was associated with minor risk of RC (HR = 0.4, p = 0.038). In conclusion, short AT is possible in selected patients with non-severe cholangitis and malignant biliary strictures. Scheduled screening for MDROs is recommended and placement of a metal stent should be performed if possible.

Active Autophagy Is Associated with Favorable Outcome in Patients with Surgically Resected Cholangiocarcinoma.

Data on the impact of autophagy in primary cholangiocarcinoma (CCA) remain scarce. Here, we therefore investigated the role of active autophagy and its impact on survival in CCA patients. All CCA patients who underwent surgical resection with curative intent between 08/2005 and 12/2021 at University Hospital Frankfurt were evaluated. Autophagic key proteins were studied by immunohistochemistry. iCCA processed for gene expression profiling of immune-exhaustion gene sets was used for an autophagy approach in silico. Active autophagy was present in 23.3% of the 172 CCA patients. Kaplan-Meier curves revealed median OS of 68.4 months (95% CI = 46.9-89.9 months) and 32.7 months (95% CI = 23.6-41.8 months) for active and non-active autophagy, respectively (p ≤ 0.001). In multivariate analysis, absence of active autophagy (HR = 2, 95% CI = 1.1-3.5, p = 0.015) was an independent risk factor for OS. Differential-expression profiling revealed significantly upregulated histone deacetylases (HDAC) mRNA in patients showing non-active autophagy. In line with this, pan-acetylated lysine was significantly more prominent in CCA patients with ongoing autophagy (p = 0.005). Our findings strengthen the role of active autophagy as a prognostically relevant marker and a potential therapeutic target.

Key role of phosphorylation sites in ATPase domain and Linker region of MLH1 for DNA binding and functionality of MutLα.

MutLα is essential for human DNA mismatch repair (MMR). It harbors a latent endonuclease, is responsible for recruitment of process associated proteins and is relevant for strand discrimination. Recently, we demonstrated that the MMR function of MutLα is regulated by phosphorylation of MLH1 at serine (S) 477. In the current study, we focused on S87 located in the ATPase domain of MLH1 and on S446, S456 and S477 located in its linker region. We analysed the phosphorylation-dependent impact of these amino acids on DNA binding, MMR ability and thermal stability of MutLα. We were able to demonstrate that phosphorylation at S87 of MLH1 inhibits DNA binding of MutLα. In addition, we detected that its MMR function seems to be regulated predominantly via phosphorylation of serines in the linker domain, which are also partially involved in the regulation of DNA binding. Furthermore, we found that the thermal stability of MutLα decreased in relation to its phosphorylation status implying that complete phosphorylation might lead to instability and degradation of MLH1. In summary, we showed here, for the first time, a phosphorylation-dependent regulation of DNA binding of MutLα and hypothesized that this might significantly impact its functional regulation during MMR in vivo.

Bone Marrow Assessment in Liver Cirrhosis Patients with Otherwise Unexplained Peripheral Blood Cytopenia.

We performed a retrospective single-center analysis to investigate the diagnostic yield of bone marrow puncture in patients with liver cirrhosis and cytopenia. Liver cirrhosis patients receiving bone marrow aspiration or biopsy for the diagnostic work-up of otherwise unexplained peripheral blood cytopenia at our institution between 2004 and 2020 were enrolled in this study. We evaluated findings from cytologic, histologic and immunologic assessment and final diagnostic outcomes. A total of 118 patients with a median age of 55 years and a median Child-Pugh score of B (8 points) were enrolled. The main etiologies of liver cirrhosis were viral hepatitis (B and C) or chronic alcohol consumption. The majority of patients (60%) exhibited concurrent anemia, leukocytopenia and thrombocytopenia. Bone marrow assessment revealed normal, unspecific or reactive alterations in 117 out of 118 patients (99%). One patient was diagnosed with myelodysplastic syndrome. Our findings suggest that peripheral blood cytopenia in patients with liver cirrhosis is rarely associated with a primary bone marrow pathology.

Pathogenetic mechanisms and therapeutic approaches of acute-to-chronic liver failure.

Liver cirrhosis is the end stage of all chronic liver diseases and contributes significantly to overall mortality of 2% globally. The age-standardized mortality from liver cirrhosis in Europe is between 10 and 20% and can be explained by not only the development of liver cancer but also the acute deterioration in the patient's overall condition. The development of complications including accumulation of fluid in the abdomen (ascites), bleeding in the gastrointestinal tract (variceal bleeding), bacterial infections, or a decrease in brain function (hepatic encephalopathy) define an acute decompensation that requires therapy and often leads to acute-on-chronic liver failure (ACLF) by different precipitating events. However, due to its complexity and organ-spanning nature, the pathogenesis of ACLF is poorly understood, and the common underlying mechanisms leading to the development of organ dysfunction or failure in ACLF are still elusive. Apart from general intensive care interventions, there are no specific therapy options for ACLF. Liver transplantation is often not possible in these patients due to contraindications and a lack of prioritization. In this review, we describe the framework of the ACLF-I project consortium funded by the Hessian Ministry of Higher Education, Research and the Arts (HMWK) based on existing findings and will provide answers to these open questions.

Risk Factors and Role of Antibiotic Prophylaxis for Wound Infections after Percutaneous Endoscopic Gastrostomy.

BACKGROUND AND STUDY AIM: The incidence of wound infections after percutaneous endoscopic gastrostomy (PEG) varies widely in recent studies. The present study systematically investigates the underlying risk factors for the development of wound infections in a large cohort of patients over a long-term follow-up period. PATIENTS AND METHODS: A retrospective cohort study of patients undergoing PEG insertion using either the pull or push technique was conducted and patients followed up for 3 years. Tube-related wound infections were identified, and pathogens regularly cultured from wound swabs. Adjusted analysis was performed via univariate and multivariate logistic regression analysis. RESULTS: 616 patients were included in this study. A total of 25% percent of patients developed wound infections upon PEG tube insertion and 6.5% showed recurrent infections. Nicotine abuse (p = 0.01), previous ischemic stroke (p = 0.01) and head and neck cancer (p < 0.001) showed an increased risk for wound infection after PEG placement. Moreover, radio-chemotherapy was associated with the occurrence of wound infections (p < 0.001). Infection rates were similar between pull and push cohorts. The most common bacterial pathogen detected was Enterobacterales (19.2%). Staphylococcus aureus, Pseudomonas aeruginosa and enterococci were frequently detected in recurrent infection (14.2%, 11.4% and 9.6%, respectively). Antibiotic prophylaxis showed no effect on infection rates. CONCLUSIONS: Wound infections after PEG placement are common and occasionally occur as recurrent infections. There is potential for improvement in everyday clinical practice, particularly regarding antibiotic prophylaxis in accordance with guidelines.