Gut microbiota predicts severity and reveals novel metabolic signatures in acute pancreatitis.

OBJECTIVE: Early disease prediction is challenging in acute pancreatitis (AP). Here, we prospectively investigate whether the microbiome predicts severity of AP (Pancreatitis-Microbiome As Predictor of Severity; P-MAPS) early at hospital admission. DESIGN: Buccal and rectal microbial swabs were collected from 424 patients with AP within 72 hours of hospital admission in 15 European centres. All samples were sequenced by full-length 16S rRNA and metagenomic sequencing using Oxford Nanopore Technologies. Primary endpoint was the association of the orointestinal microbiome with the revised Atlanta classification (RAC). Secondary endpoints were mortality, length of hospital stay and severity (organ failure >48 hours and/or occurrence of pancreatic collections requiring intervention) as post hoc analysis. Multivariate analysis was conducted from normalised microbial and corresponding clinical data to build classifiers for predicting severity. For functional profiling, gene set enrichment analysis (GSEA) was performed and normalised enrichment scores calculated. RESULTS: After data processing, 411 buccal and 391 rectal samples were analysed. The intestinal microbiome significantly differed for the RAC (Bray-Curtis, p value=0.009), mortality (Bray-Curtis, p value 0.006), length of hospital stay (Bray-Curtis, p=0.009) and severity (Bray-Curtis, p value=0.008). A classifier for severity with 16 different species and systemic inflammatory response syndrome achieved an area under the receiving operating characteristic (AUROC) of 85%, a positive predictive value of 67% and a negative predictive value of 94% outperforming established severity scores. GSEA revealed functional pathway units suggesting elevated short-chain fatty acid (SCFA) production in severe AP. CONCLUSIONS: The orointestinal microbiome predicts clinical hallmark features of AP, and SCFAs may be used for future diagnostic and therapeutic concepts. TRIAL REGISTRATION NUMBER: NCT04777812.

Time to Deliver on Promises: The Role of ERBB2 Alterations as Treatment Options for Colorectal Cancer Patients in the Era of Precision Oncology.

Receptor tyrosine kinase erythroblastic oncogene B2 (ERBB2), also known as human epidermal growth factor receptor 2 (HER2), represents an oncogenic driver and has been effectively targeted in breast and gastric cancer. Recently, next-generation sequencing (NGS) discovered ERBB2 as a promising therapeutic target in metastatic colorectal cancer (mCRC), where it is altered in 3-5% of patients, but no therapies are currently approved for this use. Herein, we present the experience of a single center in diagnosing actionable genetic ERBB2 alterations using NGS and utilizing the latest therapeutic options. Between October 2019 and December 2022, a total of 107 patients with advanced CRC underwent molecular analysis, revealing actionable ERBB2 mutations in two patients and ERBB2 amplifications in two other patients. These findings correlated with immunohistochemical (IHC) staining. Of these four patients, two were treated with trastuzumab-deruxtecan (T-DXd). We present two exemplary cases of patients with actionable ERBB2 alterations to demonstrate the effectiveness of T-DXd in heavily pretreated ERBB2-positive mCRC patients and the need for early molecular profiling. To fully exploit the potential of this promising treatment, earlier molecular profiling and the initiation of targeted therapies are essential.

Activity of acute pancreatitis is modified by secreted protein acidic and rich in cysteine ablation.

BACKGROUND: Acute pancreatitis (AP) is a frequent cause for hospitalization. However, molecular determinants that modulate severity of experimental pancreatitis are only partially understood. OBJECTIVE: To investigate the role of secreted protein acidic and rich in cysteine (SPARC) during cerulein-induced AP in mice. METHODS: AP was induced by repeated cerulein injections in SPARC knock-out mice (SPARC-/- ) and control littermates (SPARC+/+ ). Secreted protein acidic and rich in cysteine expression and severity of AP were determined by histopathological scoring, immunohistochemistry, and biochemical assays. For functional analysis, primary murine acinar cell cultures with subsequent amylase release assays were employed. Proteome profiler assay and ELISA were conducted from pancreatic tissue lysates, and co-immunofluorescence was performed. RESULTS: Upon cerulein induction, SPARC expression was robustly induced in pancreatic stellate cells (PSCs) but not in acinar cells. Genetic SPARC ablation resulted in attenuated severity of AP with significantly reduced levels of pancreatic necrosis, apoptosis, immune cell infiltration, and reduced fibrosis upon chronic stimulation. However, the release of amylase upon cerulein stimulation in primary acinar cell culture from SPARC+/+ and SPARC-/- was indistinguishable. Notably, immune cell derived C-C Motif Chemokine Ligand 2 (CCL2) was highly elevated in SPARC+/+ pancreatic tissue potentially linking PSC derived SPARC with CCL2 induction in AP. CONCLUSION: SPARC mediates the severity of AP. The potential link between SPARC and the CCL2 axis could open new avenues for tailored therapeutic interventions in AP patients and warrants further investigations.

Depletion of Macrophages Improves Therapeutic Response to Gemcitabine in Murine Pancreas Cancer.

BACKGROUND: The tumor microenvironment (TME) is composed of fibro-inflammatory cells and extracellular matrix (ECM) components. However, the exact contribution of the various TME compartments towards therapeutic response is unknown. Here, we aim to dissect the specific contribution of tumor-associated macrophages (TAMs) towards drug delivery and response in pancreatic ductal adenocarcinoma (PDAC). METHODS: The effect of gemcitabine was assessed in human and murine macrophages, human pancreatic stellate cells (hPSCs), and tumor cells (L3.6pl, BxPC3 and KPC) in vitro. The drug metabolism of gemcitabine was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Preclinical studies were conducted using KrasG12D;p48-Cre and KrasG12D;p53172H;Pdx-Cre mice to investigate gemcitabine delivery at different stages of tumor progression and upon pharmacological TAM depletion. RESULTS: Gemcitabine accumulation was significantly increased in murine PDAC tissue compared to pancreatic intraepithelial neoplasia (PanIN) lesions and healthy control pancreas tissue. In vitro, macrophages accumulated and rapidly metabolized gemcitabine resulting in a significant drug scavenging effect for gemcitabine. Finally, pharmacological TAM depletion enhanced therapeutic response to gemcitabine in tumor-bearing KPC mice. CONCLUSION: Macrophages rapidly metabolize gemcitabine in vitro, and pharmacological depletion improves the therapeutic response to gemcitabine in vivo. Our study supports the notion that TAMs might be a promising therapeutic target in PDAC.

Microenvironmental Determinants of Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) belongs to the most lethal solid tumors in humans. A histological hallmark feature of PDAC is the pronounced tumor microenvironment (TME) that dynamically evolves during tumor progression. The TME consists of different non-neoplastic cells such as cancer-associated fibroblasts, immune cells, endothelial cells, and neurons. Furthermore, abundant extracellular matrix components such as collagen and hyaluronic acid as well as matricellular proteins create a highly dynamic and hypovascular TME with multiple biochemical and physical interactions among the various cellular and acellular components that promote tumor progression and therapeutic resistance. In recent years, intensive research efforts have resulted in a significantly improved understanding of the biology and pathophysiology of the TME in PDAC, and novel stroma-targeted approaches are emerging that may help to improve the devastating prognosis of PDAC patients. However, none of anti-stromal therapies has been approved in patients so far, and there is still a large discrepancy between multiple successful preclinical results and subsequent failure in clinical trials. Furthermore, recent findings suggest that parts of the TME may also possess tumor-restraining properties rendering tailored therapies even more challenging.

Preclinical Evaluation of 1,2-Diamino-4,5-Dibromobenzene in Genetically Engineered Mouse Models of Pancreatic Cancer.

Background: Pancreatic ductal adenocarcinoma (PDAC) is highly resistant to standard chemo- and radiotherapy. Recently, a new class of non-platinum-based halogenated molecules (called FMD compounds) was discovered that selectively kills cancer cells. Here, we investigate the potential of 1,2-Diamino-4,5-dibromobenzene (2Br-DAB) in combination with standard chemotherapy and radiotherapy in murine and human PDAC. Methods: Cell viability and colony formation was performed in human (Panc1, BxPC3, PaTu8988t, MiaPaCa) and three murine LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre (KPC) pancreatic cancer cell lines. In vivo, preclinical experiments were conducted in LSL-KrasG12D/+;p48-Cre (KC) and KPC mice using 2Br-DAB (7 mg/kg, i.p.), +/- radiation (10 × 1.8 Gy), gemcitabine (100 mg/kg, i.p.), or a combination. Tumor growth and therapeutic response were assessed by high-resolution ultrasound and immunohistochemistry. Results: 2Br-DAB significantly reduced cell viability in human and murine pancreatic cancer cell lines in a dose-dependent manner. In particular, colony formation in human Panc1 cells was significantly decreased upon 25 µM 2Br-DAB + radiation treatment compared with vehicle control (p = 0.03). In vivo, 2Br-DAB reduced tumor frequency in KC mice. In the KPC model, 2Br-DAB or gemcitabine monotherapy had comparable therapeutic effects. Furthermore, the combination of gemcitabine and 2Br-DAB or 2Br-DAB and 18 Gy irradiation showed additional antineoplastic effects. Conclusions: 2Br-DAB is effective in killing pancreatic cancer cells in vitro. 2Br-DAB was not toxic in vivo, and additional antineoplastic effects were observed in combination with irradiation.

Utilizing High Resolution Ultrasound to Monitor Tumor Onset and Growth in Genetically Engineered Pancreatic Cancer Models.

The LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre (KPC) mouse model represents an established and frequently used transgenic model to evaluate novel therapies in pancreatic cancer. Tumor onset is variable in the KPC model between 8 weeks and several months. Therefore, non-invasive imaging tools are required to screen for tumor onset and monitor for response to treatment. To address this issue, different approaches have emerged over the last years. High resolution ultrasound has major advantages such as non-invasiveness, fast session times and a high image resolution without radiation exposure. However, ultrasound in mice is not trivial and sufficient anatomical knowledge and practical skills are required to successfully perform high resolution ultrasound in preclinical pancreatic cancer models. With the following article, a detailed hands-on guide for abdominal ultrasound in murine models with a particular focus on endogenous pancreatic cancer models is presented. Furthermore, a summary of common mistakes and how to avoid them is provided.