An Adaptable Protocol to Generate a Murine Enteroid-Macrophage Co-Culture System.

Impairment of the intestinal epithelial barrier is frequently seen as collateral damage in various local and systemic inflammatory conditions. The inflammatory process is characterized by reciprocal interactions between the host intestinal epithelium and mucosal innate immune cells, e.g., macrophages. This article provides step-by-step instructions on how to set up a murine enteroid-macrophage co-culture by culturing cellular elements in proximity separated by a porous membrane. Unlike previously published co-culture systems, we have combined enteroids grown from C57BL6j mice with syngeneic bone marrow-derived macrophages to preclude potential allo-reactions between immune cells and epithelium. Transformation of intestinal crypts into proliferative enteroids was achieved by cultivation in Wnt3a-Noggin-R-Spondin-conditioned medium supplemented with ROCK inhibitor Y-27632. The differentiated phenotype was promoted by the use of the Wnt3-deprived EGF-Noggin-R-Spondin medium. The resulting co-culture of primary cells can be employed as a basic model to better understand the reciprocal relationship between intestinal epithelium and macrophages. It can be used for in vitro modelling of mucosal inflammation, mimicked by stimulation of macrophages either while being in co-culture or before being introduced into co-culture, to simulate enterogenic sepsis or systemic conditions affecting the intestinal tract.

Prediction of resistance to bevacizumab plus FOLFOX in metastatic colorectal cancer-Results of the prospective multicenter PERMAD trial.

BACKGROUND: Anti-vascular endothelial growth factor (VEGF) monoclonal antibodies (mAbs) are widely used for tumor treatment, including metastatic colorectal cancer (mCRC). So far, there are no biomarkers that reliably predict resistance to anti-VEGF mAbs like bevacizumab. A biomarker-guided strategy for early and accurate assessment of resistance could avoid the use of non-effective treatment and improve patient outcomes. We hypothesized that repeated analysis of multiple cytokines and angiogenic growth factors (CAFs) before and during treatment using machine learning could provide an accurate and earlier, i.e., 100 days before conventional radiologic staging, prediction of resistance to first-line mCRC treatment with FOLFOX plus bevacizumab. PATIENTS AND METHODS: 15 German and Austrian centers prospectively recruited 50 mCRC patients receiving FOLFOX plus bevacizumab as first-line treatment. Plasma samples were collected every two weeks until radiologic progression (RECIST 1.1) as determined by CT scans performed every 2 months. 102 pre-selected CAFs were centrally analyzed using a cytokine multiplex assay (Luminex, Myriad RBM). RESULTS: Using random forests, we developed a predictive machine learning model that discriminated between the situations of "no progress within 100 days before radiological progress" and "progress within 100 days before radiological progress". We could further identify a combination of ten out of the 102 CAF markers, which fulfilled this task with 78.2% accuracy, 71.8% sensitivity, and 82.5% specificity. CONCLUSIONS: We identified a CAF marker combination that indicates treatment resistance to FOLFOX plus bevacizumab in patients with mCRC within 100 days prior to radiologic progress.

Nanoliposomal Irinotecan With Fluorouracil and Leucovorin or Gemcitabine Plus Cisplatin in Advanced Cholangiocarcinoma: A Phase II Study of the AIO Hepatobiliary-YMO Cancer Groups (NIFE-AIO-YMO HEP-0315).

PURPOSE: First-line therapy options in advanced cholangiocarcinoma (CCA) are based on the ABC-02 trial regimen (gemcitabine/cisplatin [G/C]). The NIFE trial examined nanoliposomal irinotecan/fluorouracil/leucovorin (nal-IRI/FU/LV) as alternative first-line therapy in advanced CCA. METHODS: NIFE is a prospective, open-label, randomized, multicenter phase II study that aimed at detecting efficacy comparable with the standard treatment. Patients with advanced CCA were randomly assigned (1:1) to receive nal-IRI/FU/LV (arm A) or G/C (arm B). Stratification parameters were intrahepatic versus extrahepatic CCA, sex, and Eastern Cooperative Oncology Group (ECOG; 0/1). Arm A was designed as a Simon's optimal two-stage design and arm B served as a randomized control group. The primary goal was to exclude an inferior progression-free survival (PFS) at 4 months of only 40%, while assuming a rate of 60% on G/C population. RESULTS: Between 2018 and 2020, overall 91 patients were randomly assigned to receive nal-IRI/FU/LV (n = 49) or G/C (n = 42). The NIFE trial formally met its primary end point with a 4-month PFS rate of 51% in patients receiving nal-IRI/FU/LV. The median PFS was 6 months (2.4-9.6) in arm A and 6.9 months (2.5-7.9) in arm B. Median overall survival (OS) was 15.9 months (10.6-20.3) in arm A and 13.6 months (6.5-17.7) in arm B. The exploratory comparison of study arms suggested a numerical but statistically not significant advantage for nal-IRI/FU/LV (hazard ratio for PFS, 0.85 [95% CI, 0.53 to 1.38] and for OS, 0.94 [95% CI, 0.58 to 1.50]). Analysis for stratification parameters revealed no differences for sex and ECOG, but for tumor localization. The objective response rate was 24.5% with nal-IRI/FU/LV and 11.9% with G/C. No unexpected toxicities occurred. AEs related to nal-IRI/FU/LV were mainly GI and to G/C hematologic. CONCLUSION: Treatment of advanced CCA with nal-IRI/FU/LV demonstrated efficacy in first-line therapy without new safety findings and merits further validation.

Efficiency and safety of nasal positive airway pressure systems during endoscopic procedures in high-risk patients: Endo-Breath study.

Background and study aims Sedation of high-risk patients is a relevant issue in interventional endoscopy. This is especially because standard oximetric monitors display only hypoxia and not the preceding hypercapnia. Therefore, the question arises whether use of a nasal positive airway pressure (nPAP) system can decrease the rate of sedation-associated events. Patients and methods A randomized, prospective trial was conducted at University Hospital Ulm, including 98 consecutive patients, identified as high-risk (American Society of Anesthesiologists physical status ≥3) and scheduled for prolonged (>15 minutes) endoscopic procedures. Patients underwent 1:1 randomization to two groups: interventional (nPAP-Mask) and control (conventional oxygen supplementation). Levels of CO 2 were measured noninvasively by transcutaneous capnometry device. The primary outcome was incidence of hypoxia (SpO 2 <90% over 10 seconds) and incidence of severe hypoxia was incidence of SpO 2 <80% over 10 seconds. One of our secondary objectives was to determine if the nPAP-Mask could result in significant CO 2 retention among high-risk patients. Results Data analysis showed lower incidence of hypoxia in the interventional group (10/47 vs. 31/251) P <0.05. Episodes of severe hypoxia (SpO 2 <80% over 10 seconds) were more frequent in the control group (8/51) compared with the intervention group (2/47) P <0.05. There was no significant difference in ΔCO 2 levels in the interventional vs. control group (-6.01±7.66 vs. -7.35±8.59 mm Hg). Conclusions In high-risk patients use of a nasal positive airway pressure system could significantly lower risk of hypoxia, especially in prolonged procedures. The nPAP-Mask does not induce CO 2 retention when compared with conventional oxygen supplementation.

A pancreatic cancer organoid-in-matrix platform shows distinct sensitivities to T cell killing.

Poor treatment responses of pancreatic ductal adenocarcinoma (PDAC) are in large part due to tumor heterogeneity and an immunosuppressive desmoplastic tumor stroma that impacts interactions with cells in the tumor microenvironment (TME). Thus, there is a pressing need for models to probe the contributions of cellular and noncellular crosstalk. Organoids are promising model systems with the potential to generate a plethora of data including phenotypic, transcriptomic and genomic characterization but still require improvements in culture conditions mimicking the TME. Here, we describe an INTERaction with Organoid-in-MatriX ("InterOMaX") model system, that presents a 3D co-culture-based platform for investigating matrix-dependent cellular crosstalk. We describe its potential to uncover new molecular mechanisms of T cell responses to murine KPC (LSL-KrasG12D/+27/Trp53tm1Tyj/J/p48Cre/+) PDAC cells as well as PDAC patient-derived organoids (PDOs). For this, a customizable matrix and homogenously sized organoid-in-matrix positioning of cancer cells were designed based on a standardized agarose microwell chip array system and established for co-culture with T cells and inclusion of stromal cells. We describe the detection and orthogonal analysis of murine and human PDAC cell populations with distinct sensitivity to T cell killing that is corroborated in vivo. By enabling both identification and validation of gene candidates for T cell resistance, this platform sets the stage for better mechanistic understanding of cancer cell-intrinsic resistance phenotypes in PDAC.

[Palliative Treatment of Metastatic Rectal Cancer]. / Palliative Systemtherapie des metastasierten Rektumkarzinoms.

Palliative treatment of metastatic rectal cancer (mCRC) has developed considerably in recent years, with new therapeutic strategies such as induction and maintenance therapies, the establishment of targeted therapies and molecularly defined strategies in defined subgroups such as MSI-H-patients. The following article presents evidence based therapeutic options and algorithms.

Emerging Roles of Small Extracellular Vesicles in Gastrointestinal Cancer Research and Therapy.

Discovered in the late eighties, sEVs are small extracellular nanovesicles (30-150 nm diameter) that gained increasing attention due to their profound roles in cancer, immunology, and therapeutic approaches. They were initially described as cellular waste bins; however, in recent years, sEVs have become known as important mediators of intercellular communication. They are secreted from cells in substantial amounts and exert their influence on recipient cells by signaling through cell surface receptors or transferring cargos, such as proteins, RNAs, miRNAs, or lipids. A key role of sEVs in cancer is immune modulation, as well as pro-invasive signaling and formation of pre-metastatic niches. sEVs are ideal biomarker platforms, and can be engineered as drug carriers or anti-cancer vaccines. Thus, sEVs further provide novel avenues for cancer diagnosis and treatment. This review will focus on the role of sEVs in GI-oncology and delineate their functions in cancer progression, diagnosis, and therapeutic use.

Symptoms and Management of Aseptic Liver Abscesses.

Aseptic liver abscesses occur very rarely. Clinical guidelines on the management of the disease do not exist, and the diagnosis is challenging.We screen MEDLINE and PUBMED databases for relevant case reports from inception to November 2022. Information on patient age, sex, initial symptoms, the extent of abscess formation, further diagnoses, treatment, and course of the disease is analyzed.Thirty cases with sterile hepatic abscess formation are identified. In most patients (n=18), the spleen is affected as well. Patients typically present with fever, abdominal pain, and increased inflammatory values. Comorbidity with inflammatory bowel disease is very common (n=18) and is associated with a significantly younger age at the time of hepatic abscess development. In addition, many patients show autoimmune-mediated cutaneous, ocular, or arthritic rheumatoid manifestations. Histological examination of abscess material reveals neutrophilic infiltration. The majority of patients initially receive corticosteroid therapy. Furthermore, response to azathioprine, anti-TNF-α antibodies, and other immunomodulatory drugs is reported. Ten out of fourteen patients with a long-term follow-up (≥ 36 months) have at least one relapse of hepatic abscess formation.Aseptic hepatic abscesses should be considered in the case of sterile punctures and non-response to antibiotics. Patients with aseptic liver abscesses have a high risk of recurrence warranting immunomodulatory maintenance therapy.

Protocol of the IntenSify-Trial: An open-label phase I trial of the CYP3A inhibitor cobicistat and the cytostatics gemcitabine and nab-paclitaxel in patients with advanced stage or metastatic pancreatic ductal adenocarcinoma to evaluate the combination's pharmacokinetics, safety, and efficacy.

Expression of CYP3A5 protein is a basal and acquired resistance mechanism of pancreatic ductal adenocarcinoma cells conferring protection against the CYP3A and CYP2C8 substrate paclitaxel through metabolic degradation. Inhibition of CYP3A isozymes restores the cells sensitivity to paclitaxel. The combination of gemcitabine and nab-paclitaxel is an established regimen for the treatment of metastasized or locally advanced inoperable pancreatic cancer. Cobicistat is a CYP3A inhibitor developed for the pharmacoenhancement of protease inhibitors. The addition of cobicistat to gemcitabine and nab-paclitaxel may increase the antitumor effect. We will conduct a phase I dose escalation trial with a classical 3 + 3 design to investigate the safety, tolerability, and pharmacokinetics (PKs) of gemcitabine, nab-paclitaxel, and cobicistat. Although the doses of gemcitabine (1000 mg/m2 ) and cobicistat (150 mg) are fixed, three dose levels of nab-paclitaxel (75, 100, and 125 mg/m2 ) will be explored to account for a potential PK drug interaction. After the dose escalation phase, we will set the recommended dose for expansion (RDE) and treat up to nine patients in an expansion part of the trial. The trial is registered under the following identifiers EudraCT-Nr. 2019-001439-29, drks.de: DRKS00029409, and ct.gov: NCT05494866. Overcoming resistance to paclitaxel by CYP3A5 inhibition may lead to an increased efficacy of the gemcitabine and nab-paclitaxel regimen. Safety, efficacy, PK, and RDE data need to be acquired before investigating this combination in a large-scale clinical study.

Risks and benefits of TIPS in HCC and other liver malignancies: a literature review.

BACKGROUND: Transjugular intrahepatic portosystemic shunt (TIPS) is a well-validated treatment option for clinically significant portal hypertension (CSPH) in the context of liver cirrhosis. Its high efficacy and safety in the management of treatment-refractory ascites and variceal bleeding have been extensively proven. Contraindications for TIPS include severe right heart failure, hepatic encephalopathy, and sepsis. However, the role of liver malignancy in TIPS is debatable. Mostly, primary liver malignancies such as hepatocellular carcinoma (HCC) emerge from advanced liver diseases. Coexisting portal hypertension in HCC often results in limited treatment options and a poor prognosis. Previous studies have shown that TIPS implantation in patients with HCC is technically feasible and is usually not associated with major adverse events. Furthermore, TIPS may help in bridging the time to liver transplantation in early HCC and allow for locoregional treatment in advanced HCC. However, several studies suggest that seeding tumour cells to the lungs by TIPS placement might worsen the prognosis. CONCLUSIONS: TIPS placement in patients with coexisting liver malignancy remains a case-by-case decision, and there is no profound evidence allowing general recommendations. This review aims to provide a state-of-the-art overview of the potential risks and benefits of TIPS placement in patients with liver malignancies.

Multicentric pilot study to standardize clinical whole exome sequencing (WES) for cancer patients.

A growing number of druggable targets and national initiatives for precision oncology necessitate broad genomic profiling for many cancer patients. Whole exome sequencing (WES) offers unbiased analysis of the entire coding sequence, segmentation-based detection of copy number alterations (CNAs), and accurate determination of complex biomarkers including tumor mutational burden (TMB), homologous recombination repair deficiency (HRD), and microsatellite instability (MSI). To assess the inter-institution variability of clinical WES, we performed a comparative pilot study between German Centers of Personalized Medicine (ZPMs) from five participating institutions. Tumor and matched normal DNA from 30 patients were analyzed using custom sequencing protocols and bioinformatic pipelines. Calling of somatic variants was highly concordant with a positive percentage agreement (PPA) between 91 and 95% and a positive predictive value (PPV) between 82 and 95% compared with a three-institution consensus and full agreement for 16 of 17 druggable targets. Explanations for deviations included low VAF or coverage, differing annotations, and different filter protocols. CNAs showed overall agreement in 76% for the genomic sequence with high wet-lab variability. Complex biomarkers correlated strongly between institutions (HRD: 0.79-1, TMB: 0.97-0.99) and all institutions agreed on microsatellite instability. This study will contribute to the development of quality control frameworks for comprehensive genomic profiling and sheds light onto parameters that require stringent standardization.

A Systematic Evaluation of the SARS-CoV-2 Vaccine-Induced Anti-S-RBD-Ig Response in a Population of Health Care Workers.

In the wake of the COVID-19 pandemic, the novel class of mRNA vaccines has been granted first-time approval for active immunization against SARS-CoV-2 alongside the already established viral vector-based vaccines. In this prospective single-center study, we set out to determine the vaccine-induced humoral immune response in a population of 1512 health care employees after the second and third vaccination, respectively. Anti-SARS-CoV-2 receptor-binding domain (RBD) and nucleocapsid antigen antibody concentrations were assessed using commercially available immunoassays. We could show that, in particular, young study subjects aged below 30 years, as well as those with a prior SARS-CoV-2 infection, developed significantly higher antibody concentrations. Our data further suggest that being in physically close contact with formerly SARS-CoV-2-positive people positively affects the post-vaccination response. Surprisingly, study subjects with a BMI > 30 produced the highest anti-S-RBD Ig antibody levels if they had recently received their third vaccination. Also, heterologous dual vaccine regimens consisting of a BNT162b2 and ChAdOx1 n-CoV-19, a homologous triple combination of BNT162b2, and an application of mRNA-1273 as the third vaccine, were most efficient at eliciting a humoral immune response. Our study substantiates existing evidence, but beyond that, scrutinizes the impact of vaccine agents and their respective combinations, as well as different time intervals on humoral immunogenicity.

Treatment sequences and prognostic/predictive factors in metastatic pancreatic ductal adenocarcinoma: univariate and multivariate analyses of a real-world study in Europe.

BACKGROUND: Real-world data on treatment patterns/outcomes for metastatic pancreatic cancer (mPAC) are limited. This study aims to assess real-world treatment patterns, survival outcomes, and prognostic/predictive factors in patients with mPAC. METHODS: Retrospective, observational, chart-review involving medical oncologists and gastroenterologists from five European countries. Physicians reported information on disease and patient characteristics, diagnosis, and treatment for patients diagnosed with mPAC from January-October 2016. Outcomes included median progression-free survival (mPFS), median overall survival (mOS), and the impact of baseline performance status on survival. Univariate/multivariate regression analyses were undertaken to identify prognostic/predictive factors. RESULTS: Three hundred four physicians and 3432 patients were included. First-line therapies included modified (m)FOLFIRINOX (28.4%), gemcitabine + nab-paclitaxel (28.0%), and gemcitabine monotherapy (23.0%). Frequent second-line therapies were gemcitabine monotherapy (25.0%), fluorouracil (5-FU) + oxaliplatin (21.8%), and gemcitabine + nab-paclitaxel (16.7%). Most frequent first- to second-line treatment sequences were gemcitabine + nab-paclitaxel followed by fluoropyrimidine combinations. Longest unadjusted estimated mOS was observed with (m)FOLFIRINOX followed by gemcitabine-based combinations (19.1 months). Multivariate analysis identified significant prognostic/predictive factors for OS and PFS including performance status and carbohydrate antigen 19-9 (CA 19-9) levels. CONCLUSIONS: Treatment and treatment sequences were generally in accordance with guidelines at the time of the study. Identification of prognostic/predictive factors for survival may help inform the individualised management of mPAC patients in the future.

DNA methylation-associated allelic inactivation regulates Keratin 19 gene expression during pancreatic development and carcinogenesis.

Within the pancreas, Keratin 19 (KRT19) labels the ductal lineage and is a determinant of pancreatic ductal adenocarcinoma (PDAC). To investigate KRT19 expression dynamics, we developed a human pluripotent stem cell (PSC)-based KRT19-mCherry reporter system in different genetic backgrounds to monitor KRT19 expression from its endogenous gene locus. A differentiation protocol to generate mature pancreatic duct-like organoids was applied. While KRT19/mCherry expression became evident at the early endoderm stage, mCherry signal was present in nearly all cells at the pancreatic endoderm (PE) and pancreatic progenitor (PP) stages. Interestingly, despite homogenous KRT19 expression, mCherry positivity dropped to 50% after ductal maturation, indicating a permanent switch from biallelic to monoallelic expression. DNA methylation profiling separated the distinct differentiation intermediates, with site-specific DNA methylation patterns occurring at the KRT19 locus during ductal maturation. Accordingly, the monoallelic switch was partially reverted upon treatment with a DNA-methyltransferase inhibitor. In human PDAC cohorts, high KRT19 levels correlate with low locus methylation and decreased survival. At the same time, activation of oncogenic KRASG12D signalling in our reporter system reversed monoallelic back to biallelic KRT19 expression in pancreatic duct-like organoids. Allelic reactivation was also detected in single-cell transcriptomes of human PDACs, which further revealed a positive correlation between KRT19 and KRAS expression. Accordingly, KRAS mutant PDACs had higher KRT19 mRNA but lower KRT19 gene locus DNA methylation than wildtype counterparts. KRT19 protein was additionally detected in plasma of PDAC patients, with higher concentrations correlating with shorter progression-free survival in gemcitabine/nabPaclitaxel-treated and opposing trends in FOLFIRINOX-treated patients. Apart from being an important pancreatic ductal lineage marker, KRT19 appears tightly controlled via a switch from biallelic to monoallelic expression during ductal lineage entry and is aberrantly expressed after oncogenic KRASG12D expression, indicating a role in PDAC development and malignancy. Soluble KRT19 might serve as a relevant biomarker to stratify treatment. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.