Introduction

Endoscopic retrograde cholangiopancreatography (ERCP), in surgically altered anatomy (SAA), can be challenging and the choice of the optimal technique remains debatable in clinical practice.

Aims & Methods

Aim of the study is to evaluate technical and clinical success of ERCP performed for biliopancreatic adverse events after pancreaticoduodenectomy (PD) using a wide caliber operative working channel (3.8 mm) pediatric colonoscope. It is a retrospective study conducted in a tertiary referral center. Patients who underwent ERCP between 01/02/2012 and 15/11/2022 for biliopancreatic complications after PD were included. Technical success was considered as effective endoscopic treatment of the specific complication including cannulation of pancreatico-jejunal or hepatico-jejunal anastomosis. Clinical success was considered as no need for rescue treatment (surgery or interventional radiology).

Results

During the period study, 129 patients (76 males, mean age 66 y.o., range 23-87 y.o.) underwent a total of 315 ERCP for endoscopic treatment of biliopancreatic complications after PD. The most frequent indications were biliary complications (78 patients, 60.4%, included 44 patients with anastomosis stricture, 27 patients with dehiscence and 7 patients with biliary stones), pancreatic complications (39 patients, 30.2%, included 10 patients with anastomosis strictures and 29 dehiscence) and other complications in the remaining 12 patients (6 patients with recurrence of neoplasia, 6 patients with bowel strictures). Timing between diagnosis of complication and ERCP was significantly shorter in septic patients than in the elective ones (p < 0.0001). In 78 patients (60.4%) ERCP has been performed with pediatric colonoscope; each patients underwent a mean of 2.6 ERCP (median 2 procedures). Concerning this group, anastomosis cannulation has been achieved directly in 69 patients and thanks to EUS-guided entero-enteral bypass approach in 9 patients and the procedure was performed with retrograde approach. Technical success, described as cannulation was obtained in 100% of cases. In 65/78 cases (83.3%) clinical success was obtained retrograde and in 13/78 cases (16.6%) technical success was obtained with anterograde EUS-guided approach: hepaticogastrotomy (6 cases), pancreaticogastrostomy (4 cases) and EUS-guided endoscopic drainage placement (3 cases).
Clinical success was 93,8% (121 patients). 8 patients experienced adverse events: 4 patients had recurrence of acute cholangitis and 4 patients needed rescue treatment (PTBD or surgical).

Conclusion

Endoscopic treatment for adverse events after PD achieved high technical and clinical success rates in tertiary referral centers with specific know-how in ERCP in surgically altered anatomy.

Introduction

Pancreatic ductal adenocarcinoma (PDAC) is in the tenth place of newly diagnosed case of all tumors and one of the most lethal cancer related malignancies in developed countries [1]. Surgical reduction still is the main treatment method, but often not effective due to the advanced stage. Immunotherapy is a promising treatment for many cancers. However, the efforts and attempts to use immunotherapy in the treatment of PDAC are extensive and real breakthroughs have not been achieved [2]. Peripheral blood mononuclear cells (PBMC) as immune cells are crucial for the body's response against cancer [3]. One of the main immune checkpoint member is programmed cell death protein 1 (PD1). It is known that PD1 expression in T cells is strongly regulated by aryl hydrocarbon receptor (AHR) [4]. Moreover, there are immunological barriers to treatment challenge in cancer like insufficient immune function activation and excess immune suppression [5]. Previously we analyzed immune response disbalance regards of AHR expression in PDAC patients PBMCs. Results showed significant changes of lower AHR gene expression patients' group. Next, we decided to check PBMCs functions in healthy participants after indirect interaction with pancreatic cancer cells. We hypothesized that AHR expression differences in PBMCs could regulate anti-cancer response.

Aims & Methods

The aim of this study was to analyze PBMCs functions and targets expression of healthy participants after indirect interaction with pancreatic cancer cells. PBMCs (n=11) were isolated from peripheral blood by Ficoll-Paque gradient centrifugation. Analysis was done at 3 points. The first point of study was prepared immediately after PBMCs isolation. Second and third points were prepared after 20h of PBMCs -/+ co-cultivation with BxPC3 cells. Phagocytic activity analysis was prepared after 3-hour PBMCs activation with LPS. ROS production was measured effecting with TBHP. NO production was measured after activation with LPS + L-Arginine. Simultaneously PBMCs metabolic activity was measured by resazurin assay. Monocytes and lymphocytes differentiation was evaluated by FACS analysis. Gene expressions were analyzed by RT-PCR method. Statistical analysis was performed using GraphPad software and the Mann – Whitney test.

Results

The results showed that AHR and IL1b, IL6, IL10 mRNA were increased, only IL6 was significantly upregulated after PBMC co-cultivation with BxPC3 cells. While PD1 and IL4 mRNA expression were decreased. The expression of AHR strongly negatively correlated with expression of PD1 in healthy volunteers PBMCs’ after co-cultivation with cancer cells. M1 type monocytes and NK cells decreased after co-cultivation with PDAC cells. Moreover, M0 type monocytes and cytotoxic T lymphocytes were increased. Phagocytic activity and NO production were more intensive in PBMC cells after co-cultivation. ROS production did not show significant changes.

Conclusion

This study showed negative PD1 gene expression correlation with AHR expression after indirect interactions with cancer cells in healthy participants PBMCs. Also, PBMCs co-cultivation with cancer cells initiated strong functional response and higher expression of inflammatory cytokines. These results show us different AHR relations with immunity response in healthy and cancer patients. Meanwhile, this knowledge lets us understand the importance of AHR modulation as an additional PDAC treatment strategy.

References

1. Siegel, R. L., Miller, K. D., Wagle, N. S., & Jemal, A. (2023). Cancer statistics, 2023. CA: A Cancer Journal for Clinicians, 73(1), 17-48. doi:10.3322/caac.21763
2. Mundry, C. S., Eberle, K. C., Singh, P. K., Hollingsworth, M. A., & Mehla, K. (2020). Local and systemic immunosuppression in pancreatic cancer: Targeting the stalwarts in tumor’s arsenal. Biochimica Et Biophysica Acta. Reviews on Cancer, 1874(1), 188387. doi:10.1016/j.bbcan.2020.188387
3. Griffin JF, Poruk KE, Wolfgang CL. Pancreatic cancer surgery: past, present, and future. Chin J Cancer Res. 2015 Aug;27(4):332-48
4. Liu, Y., Liang, X., Dong, W., Fang, Y., Lv, J., Zhang, T., . . . Huang, B. (2018). Tumor-repopulating cells induce PD1 expression in CD8+ T cells by transferring kynurenine and AhR activation. Cancer Cell, 33(3), 480-494.e7. doi: 10.1016/j.ccell.2018.02.005
5. Rojas, L. A., & Balachandran, V. P. (2021). Scaling the immune incline in PDAC. Nature Reviews. Gastroenterology & Hepatology, 18(7), 453-454. doi:10.1038/s41575-021-00475-9

Disclosure

The authors have no conflicts of interest to declare

Introduction

Endoscopic ultrasound-guided fine-needle biopsy (EUS-FNB) is about to become the standard procedure to distinguish solid pancreatic lesions and is typically performed using a 22G needle. Although smaller needles may be more flexible for positioning, larger needle diameters might produce bigger tissue cores and thus improve histological and molecular analysis.
The aim of this single-institution, prospective, controlled study was to compare the sample adequacy and diagnostic yield of solid pancreatic lesions using the 19G and 22G Franseen-tip EUS-FNB devices (Boston Scientific Acquire™).

Aims & Methods

The study was approved by the LMU Munich IRB and registered to German Clinical Trial Registry (DRKS00021088). Adult patients with solid pancreatic lesions on imaging (CT, MRI or ultrasound) were prospectively recruited between April 2019 and April 2022. Diagnostic EUS-FNB was performed by experienced investigators utilizing both needle sizes with 1-2 needle passes each. A blinded pathologist graded the diagnostic quality of the samples and diagnostic accuracy was calculated.

Results

The mean age at presentation was 67 years (IQR: 59 -77.5) with a ratio of 1.2:1 of male to female. Of 119 included patients, 19 had to be excluded. In 85 out of the remaining 100 patients, only one needle could be used in the lesion due to technical difficulties or complications. The final diagnoses included pancreatic cancer (68.6%), neuroendocrine tumors (4.7%), lymphoma (1.2%), extrapancreatic malignancies (11.6%), chronic pancreatitis (7.0%), autoimmune pancreatitis (2.3%) and others (4.7%). The overall EUS-guided sampling sensitivity for the 22G-needle was 74.2% and for the 19G-needle 77.2%. The diagnostic sensitivity in malignant lesions was similar in both needles (22G: 78.9%, 19G: 79.1%) and was 82.0% (22G) and 78.6% (19G) when regarding only cases with pancreatic ductal adenocarcinoma (PDAC). Post-procedural bleeding occurred in 8 cases whereas in 3 patients’ further intervention was necessary.

Conclusion

In this prospective, controlled study, the use of a 19G EUS-FNB needle did not result in a higher diagnostic accuracy of solid pancreatic lesions compared to the standard size 22G.

Disclosure

Nothing to disclose

Introduction

Gut dysbiosis reduces immunogenicity of non-COVID-19 vaccines. While immunogenicity of BNT162b2 wanes with time, factors influencing durability of vaccine immunogenicity are underinvestigated.

Aims & Methods

Aim: We aimed to investigate association between gut microbiota composition and longer-term immunogenicity of BNT162b2.
Methods: This was a prospective cohort study recruiting adult BNT162b2 two-dose recipients from three vaccination centers in Hong Kong. Exclusion criteria included prior COVID-19, gastrointestinal surgery and immunocompromised status. Blood samples were collected at baseline and day180 after first dose, and tested for neutralising antibody (NAb) against receptor-binding domain (RBD) of wild type SARS-CoV-2 virus using chemiluminescence immunoassay. NAb seroconversion (defined as >=15 AU/mL) was determined at day180. Those with NAb 75^th percentile and <75^th percentile were classified as high- and low-responders, respectively. Stool samples were collected at baseline, followed by DNA extraction and shotgun metagenomic sequencing by Illumina NovaSeq 6000 platform. Relative abundances of microbial taxa were compared between high- and low-responders. Alpha-diversity was estimated by observed species, Shannon and Simpson indices, and compared between the two groups using Wilcoxon signed-rank test. Beta-diversity was compared by non-metric multidimensional scaling (NMDS). Linear discriminant analysis (LDA)-Effect Size (LEfSe) was performed to identify stool bacterial species and metabolic functional pathways with LDA score >=2. Multivariable logistic regression adjusting for clinical factors (including age, sex, diabetes mellitus, overweight/obese) was used to derive adjusted odds ratio (aOR) of persistently high vaccine response with bacterial species.

Results

Of 242 BNT162b2 recipients (median age:50.2 years [IQR:42.5-55.6];male:85 [35.1%]), 232 (95.9%) remained seropositive at day180. 61 (25.2%) of vaccine recipients were persistent high-responders (median NAb level:205.4 AU/mL; IQR:164.6-291.3) while 181 (74.8%) were low-responders (median NAb level:40.9 AU/mL; IQR:25.8-66.4). The baseline demographics were comparable between the two groups (all p>0.05). There was no difference in the alpha and beta diversity between the two gropus (all p>0.05). Ruminococcus bicirculans, Parasutterella excrementihominis, and Streptococcus salivarius were more abundant in high- than low-responders (0.9% vs 0.1%, 0.08% vs 0.02%, 0.06% vs 0.04%, respectively), while Bacteroides thetaiotaomicron was less abundant (1.1% vs 2.1%). Consistently, LEfSe showed that R. bicirculans (log₁₀LDA score=3.65), P. excrementihominis (score=2.82), and S. salivarius (score=2.31), were enriched in persistent high-responders, while B. thetaiotaomicron was enriched in low-responders (score= –3.70). On multivariable analysis, bacterial species including R. bicirculans (aOR:1.87;95% CI:1.02-3.51), P. excrementihominis (aOR:2.20;95% CI:1.18-4.18) and S. salivarius (aOR:2.09;95% CI:1.13-3.94), but not clinical factors, were associated with persistent high response. R. bicirculans positively correlated with metabolic pathways enriched in persistent high-responders, including superpathway of L-cysteine biosynthesis (score=2.25) and L-isoleucine biosynthesis I (score =2.16), which are known to benefit immune system.

Conclusion

Potential microbial markers for longer-term BNT162b2 immunogenicity were R. bicirculans, P. excrementihominis, S. salivarius and B. thetaiotaomicron. Quantitative polymerase chain reaction (qPCR) will be performed in a validation cohort.

Disclosure

Supported by the Health and Medical Research Fund, The Government of the HKSAR (ref no: COVID1903010, Project 16)

Introduction

Pancreatic cancer is a highly lethal disease, commonly characterized by aggressiveness, affection for chemoresistance, and tumor heterogeneity. As one of the most frequent genetic alterations, mutated KRAS is described as a key driver of pancreatic tumor development and progression.

Aims & Methods

In order to gain a better understanding of KRAS-dependent signaling, we employ our recently developed stem cell-derived pancreatic ductal-like organoids (PDLOs) as an in vitro early-stage cancer model followed by a subsequent and timed single cell (sc)RNA-seq analysis. This allows a comprehensive cell characterization and the investigation of early oncogenic events at cellular resolution, aiming to identify novel clinically relevant molecular mechanisms.

Results

The scRNA-seq analysis revealed that cells expressing high levels of mutated KRAS undergo a separation into two subpopulations with distinct gene expression signatures, including overexpression of genes related to cell motility and migration as well as enhanced cytokine-related signaling. Integrating this data in a sc-resolved pancreatic cancer data set allowed ligand-receptor analysis and cross-talk studies within a virtual pancreatic cancer space with its corroborating niche. Novel pathways have been identified and validated within in vitro studies. Subsequent co-culture systems showed that KRAS^G12D-expressing PDLOs are capable of inducing cancer-associated fibroblast (CAF) differentiation as well as shielded K-ras activated yet non-cancerous organoids from T cell infiltration in vitro.

Conclusion

Based on these gained findings, it is therefore hypothesized that overexpression of mutated KRAS^G12D in healthy duct leads to the preparation of a tumor niche. In sum, we provide the evidence that oncogenic KRAS-signaling triggers niche-preparatory signals not only preceding but even allowing dysplasia to take place.

Introduction

Despite decades of extensive research, pancreatic ductal adenocarcinoma (PDAC) has been predicted to rise to the second leading cause of cancer-related death in 2030. A major hallmark of pancreatic cancer is a strong desmoplastic reaction and reduced vascularization, resulting in reduced nutrient perfusion. Although previous studies have demonstrated how the mutational landscape impacts the metabolic phenotype, assigning specific metabolic patterns to a given genotype is still of high interest for development of targeted therapeutic approaches. Homologous recombination deficiency (HRD), caused by mutations such as ATM, promotes PDAC aggressiveness, a mesenchymal phenotype, and a desmoplastic reaction.

Aims & Methods

To assess the impact of ATM deficiency driven HRD on PDAC metabolism, we investigated murine ATM-knockout PDAC cell lines. Precisely, we performed a wide range of in vitro experiments to determine cell viability, apoptosis, and functionality, of PDAC cells, as well as ATP production rates under normal and nutrient-depleted conditions. To decipher the underlying mechanisms of metabolic reprogramming in Atm-deficient PDAC cells, an individualized compound library targeting the main metabolic pathways for energy and macromolecule production was designed.

Results

Here, we show that loss of ATM facilitates the adaptation of PDAC cells to nutrient scarcity, evading metabolic stress-induced apoptosis. Mechanistically, adaptation to nutrient stress was accompanied by a higher vulnerability to inhibitors targeting ATP generating pathways oxidative phosphorylation (OxPhos) and glycolysis, as well as pentose phosphate pathway branching from glycolysis. Furthermore, especially in a metabolic stress context, Atm-deficient cells seem to rely on acetate metabolism, which is channeled into fatty acid synthesis and TCA cycle/ OxPhos. In line, loss of ATM correlated with increased ATP production. These in vitro results are supported by protein expression data in human PDAC showing a negative correlation between expression of ATM and enzymes related with OxPhos, glycolysis and acetate metabolism. Additionally, loss of ATM promotes maintenance of PDAC cell aggressive features under nutrient starvation, including a mesenchymal phenotype and DNA damage accumulation.

Conclusion

Our results indicate that specific metabolic reprogramming, affecting ATP generation and macromolecule biosynthesis pathways and occurring in ATM-deficient malignant HRD cells upon nutrient stress, promotes cell survival and tumor aggressiveness, which may ultimately contribute to cancer progression. Overall, we demonstrate how PDAC cell biology is impacted by genotype-specific metabolic remodeling, enabling exploitation of HRDness metabolic vulnerabilities through various interference strategies.

Introduction

Despite advances in cancer treatment, pancreatic adenocarcinoma continues to have a poor prognosis and remains a leading cause of death among all tumors [1]. Most pancreatic adenocarcinoma patients are diagnosed in stages with metastasis. Moreover, pancreatic adenocarcinoma has a restricted ability to respond to immunotherapy due to low immune infiltration. Prior studies have demonstrated a correlation between tumor endothelial cells (TECs) and angiogenesis, which has been indicated to play a critical role in T-cell-mediated immunosurveillance [2]. Identifying the specific subpopulation of TEC that correlates with immune evasion in pancreatic adenocarcinoma remains a challenge. However, to clarify this subpopulation may bring novel hope to patients with pancreatic adenocarcinoma.

Aims & Methods

Our study aimed to identify the specific subpopulation of TECs associated with increased immunoevasion. Analysis based on the TCGA database, scRNA-seq, the online tool Kaplan Meier plotter, and spatial transcriptomics data were utilized in our study. Moreover, we cultured human umbilical vein endothelial cells (HUVECs) and performed siRNA transfection and Western blotting to confirm the function of RASIP1 in endothelial cells.

Results

Genes related to patient survival in pancreatic adenocarcinoma were identified by the analysis from the Cancer Genome Atlas (TCGA). On the other hand, based on scRNA-seq data, the genes exclusively expressed in endothelial cells were identified. Our analysis revealed that RASIP1 (Ras Interacting Protein 1) was specifically expressed in endothelial cells and linked to improved pancreatic cancer patient survival. Subsequently, the gene ontology analysis of RASIP1-positive TECs was performed based on the scRNA-seq data, and it turned out that RASIP1-positive TECs were related to leukocyte transendothelial migration. Intriguingly, based on the analysis of the TCGA database and spatial transcriptomics data, it was revealed that RASIP1-positive TECs are correlated with increased T cell infiltration and elevated T-effector and IFN-gamma gene signatures in pancreatic adenocarcinoma. These findings suggest that RASIP1-positive TECs may contribute to the "hot" tumor phenotype by promoting T cell infiltration. To further investigate the relationship between RASIP1 and leukocyte transendothelial migration, we knocked down RASIP1 in human umbilical vein endothelial cells (HUVECs) and found a decrease in the expression of the key adhesion molecule intercellular cell adhesion molecule (ICAM). Moreover, the potential related signaling pathway was also investigated, including Erk and p38. Intriguingly, the survival analysis based on patients with pan-cancer immunotherapy also indicated the expression of RASIP1 was proportional to the survival time of cancer patients receiving immunotherapy. In the patients with high expression of RASIP1, longer survival was shown, with a p-value of 0.014 and HR of 0.74. And particularly, in patients treated with PD-L1, significantly longer survival was shown in the group with high expression of RASIP1, with a p-value of 0.01 and HR of 0.74

Conclusion

All in all, our study revealed a potential novel type of endothelial cell in pancreatic adenocarcinoma, which correlated with increased leukocyte transendothelial migration and may provide a potentially effective therapeutic mechanism.

References

1. Ullman NA, Burchard PR, Dunne RF, Linehan DC. Immunologic Strategies in Pancreatic Cancer: Making Cold Tumors Hot. J Clin Oncol. 2022 Aug 20;40(24):2789-2805.
2. Nagl L, Horvath L, Pircher A, Wolf D. Tumor Endothelial Cells (TECs) as Potential Immune Directors of the Tumor Microenvironment - New Findings and Future Perspectives. Front Cell Dev Biol. 2020 Aug 19;8:766.