Introduction

Pancreatic ductal adenocarcinoma (PDAC) is the seventh leading cause of cancer related death worldwide, with a progressive increasing incidence¹ and 5 years overall survival (OS) of 12.5% irrespective of the disease stage². Given the tumor biological aggressiveness and the limited response to chemotherapy, it arises the need to develop preclinical study models able to understand the underlying mechanism of tumor progression and chemoresistance³. These models need to be easily generable, reproducible, representative of the entire population affected by PDAC and to have the ability of mimicking the cancer behavior. Patients derived organoids (PDOs) are models of epithelial cells with self-regeneration and self-organization abilities able to behave and mimic the tumors of origin⁴. Liquid biopsy has also gained importance in tailoring the treatment for oncological patients, although the role of bile as biological fluid in patients affected by biliopancreatic malignancies is still uncertain.

Aims & Methods

We performed a prospective observational study aiming to evaluate the feasibility to create tridimensional PDOs from fine needle biopsy (FNB) samples of patients affected by PDAC and to correlate PDOs with native cancer histology. The secondary endpoint of our study was to assess the feasibility of using bile as biological fluid for cell free DNA (cfDNA) and to investigate the landscape of cfDNA in bile and plasma of patients affected by PDAC.
Patients with strong suspicion or recent diagnosis of PDAC and concomitant jaundice were included. Each patient underwent EUS guided FNB (EUS-FNB) for histological diagnosis and organoid collection was performed. By the time of biliary drainage, bile and plasma were collected for DNA extraction and analysis.

Results

From June 2023 to September 2023, 36 patients with neoplastic jaundice were evaluated. Eighteen patients with final diagnosis of PDAC have been included in the study. Eighteen underwent biliary drainage with collection of bile and plasma. Sixteen underwent EUS-FNB for organoid collection.
Mean tumor size was 33.7± 9.6 mm. Tumor was located in the head of pancreas in 94.4% of cases. Nine patients (50%) were metastatic by the time of diagnosis, eight patients (44%) had locally advanced disease and only one patient was considered resectable. Tissue acquisition was performed using FNB needles. Organoids establishment rate was successful in 100% of cases. No EUS-FNB related adverse events were observed.
At a quality concordance pathological analysis between PDAC and PDOs, morphological similarities have been observed in all cases (100%). Cellular morphology and nuclei atypia were the most consistent similarities. Organoids showed a glandular over expression compared to native PDAC. Extracted cfDNA was significantly higher in bile supernatant than in plasma (2.9 ng/ml vs 19.8 ng/ml p=0.0001). No significant differences were found for plasma and bile cfDNA among disease stages. Higher level of bile cfDNA in locally advanced disease and a trend of higher level of plasma cfDNA in metastatic stage. Bile cfDNA showed longer DNA fragment compared to plasma.

Conclusion

Organoids establishment is feasible and safe with a single passage of EUS-FNB. PDOs maintain the morphological architecture of native cancer, although the absence of dense stromal reaction leads to a remarkable organoids ability to create glandular structures. Qualitative and quantitative composition of ctDNA is superior in bile rather than in plasma, making this fluid an important tool for NGS analysis and a better comprehension of tumor mutations.

References

1. Sung H, Ferlay J, Siegel RL, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin. 2021;71(3):209-249. doi:10.3322/caac.21660
2. Arnold M, Rutherford MJ, Bardot A, et al. Progress in cancer survival, mortality, and incidence in seven high-income countries 1995-2014 (ICBP SURVMARK-2): a population-based study. Lancet Oncol. 2019;20(11):1493-1505. doi:10.1016/S1470-2045(19)30456-5
3. Baker LA, Tiriac H, Clevers H, Tuveson DA. Modeling pancreatic cancer with organoids. Trends Cancer. 2016;2(4):176-190. doi:10.1016/j.trecan.2016.03.004
4. Moreira L, Bakir B, Chatterji P, Dantes Z, Reichert M, Rustgi AK. Pancreas 3D Organoids: Current and Future Aspects as a Research Platform for Personalized Medicine in Pancreatic Cancer. Cell Mol Gastroenterol Hepatol. 2017;5(3):289-298. Published 2017 Dec 16. doi:10.1016/j.jcmgh.2017.12.004

Introduction

Low Phospholipid-Associated Cholelithiasis (LPAC) syndrome is a rare disorder caused by mutations in the ABCB4 phospholipid transporter, found in biliary canaliculi. These mutations lead to a deficiency in phospholipids and the subsequent precipitation of cholesterol crystals within the small intrahepatic bile ducts. LPAC syndrome can be diagnosed following the emergence of biliary symptoms or through familial screening. The objective of our study is to characterize the epidemiological, clinical, and paraclinical data of patients monitored for LPAC syndrome in our institution and to assess therapeutic responses and outcomes.

Aims & Methods

We collected data from all patients diagnosed with LPAC syndrome at our institution. Diagnosis was established based on the presence of at least two of the following criteria: (1) age at onset of biliary symptoms ≤40 years; (2) presence of hyperechoic intrahepatic foci ("comet tails" or micro-spots); (3) recurrence of biliary symptoms post-cholecystectomy.

Results

10 patients were included. The Average age at diagnosis was 33,6 years, with symptoms typically beginning around age 30. The sex ratio was 2/8 (M/F).a family history of biliary lithiasis and cholecystectomy before the age of 40 were found in 70% of cases . None of the female patients had a history of pregnancy-related cholestasis. 70% of the patients had undergone cholecystectomy prior to their LPAC diagnosis, with recurrence of biliary symptoms before age 40 in all these patients. Clinical presentations upon admission included symptomatic gallstone disease in 5 cases, acute cholangitis due to main bile duct stones in 4 cases, and acute lithiasic pancreatitis in 1 case. Hepatobiliary ultrasound was performed in all patients, revealing comet tail signs in 50% of cases, hyperechoic intrahepatic spots in 20% of cases, and intrahepatic stones in 50% of cases. Biochemical tests showed hyperbilirubinemia in 30% of cases, elevated Gamma-GT and alkaline phosphatase in 80% of cases, and hepatocellular damage in 40% of cases. A mutation in the ABCB4 gene was found in 2-patients.8 patients benefit from An Ursodeoxycholic Acid (UDCA) dosage averaging 11mg/kg, experiencing significant clinical and biological improvements.

Conclusion

LPAC syndrome is a rare pathology in our series, predominantly affecting young, non-overweight women with personal and family history of early biliary stones occurrence . Treatment with Ursodeoxycholic Acid (UDCA) controlled symptoms in the majority of cases.

Clinical Case Summary

A 23-year-old female presented with epigastric pain, jaundice, and elevated liver enzymes. Imaging confirmed a mildly dilated common bile duct (CBD) with multiple filling defects. ERCP revealed a normal ampulla and a half-sphincterotomy was performed with three stones retrieved via balloon trawl. The procedure was uneventful, and she was discharged the same day.
Three days later, she re-presented with dizziness, melaena, and a haemoglobin drop from 127 g/L to 85 g/L. Gastroscopy demonstrated active oozing from the sphincterotomy site, managed successfully with placement of a fully covered metal stent (FCMS). She remained stable and was discharged with outpatient follow-up.
Three weeks later, she re-presented with partial stent blockage and underwent repeat ERCP for stent removal and duct clearance. Within 24 hours, she developed acute left shoulder tip and chest pain. Cardiac investigations were negative. CT imaging demonstrated a large haemoperitoneum and a bulging splenic haematoma without active bleeding, confirmed on a diagnostic laparoscopy .
She was managed conservatively in intensive care and follow up imaging confirmed complete resolution of the haematoma.
This case illustrates splenic haematoma as a rare yet significant complication of ERCP, with only a handful of cases described in the literature. Proposed mechanisms involve shear forces transmitted via the splenogastric ligament, typically in patients with known risk factors such as intra-abdominal adhesions, previous surgery, chronic pancreatitis, or cirrhosis. However, this case highlights its occurrence in a young, healthy patient with no risk factors, suggesting procedural technique or anatomical variation to be a potential trigger for injury. Symptoms such as shoulder or upper quadrant pain following ERCP should prompt consideration of splenic injury. Early recognition is key, as many cases resolve with conservative management but may carry a risk of life-threatening bleeding if unrecognised.

References

MRCP Image Demonstrating CBD Filling Defects
Coronal MRCP showing a mildly dilated common bile duct with multiple signal voids consistent with choledocholithiasis.
ERCP Image Showing Duct Clearance
Fluoroscopic image during ERCP showing balloon trawl and retrieval of multiple CBD stones following half-sphincterotomy.
Blood Results Demonstrating Haemoglobin Drop
Laboratory panel showing haemoglobin decline from 127 g/L to 85 g/L following gastrointestinal bleeding.
Gastroscopy Image Demonstrating Oozing and FCMS Placement
Endoscopic image showing active oozing at the sphincterotomy site and successful placement of a fully covered metal stent.
CT Image of Splenic Haematoma
Axial CT abdomen showing extensive haemoperitoneum and a bulging splenic haematoma in the left upper quadrant.
Follow-up CT Confirming Resolution
Interval CT imaging showing complete resolution of the previously identified splenic haematoma.

Introduction

Microbiome analysis is a promising approach for the application of findings from basic research in the clinical field. However, there is an ongoing debate about the consistency of different bioinformatics analysis platforms. The lack of established standards affects the implementation of research findings.

Aims & Methods

In our study, we demonstrated that the results on the microbial composition of gastric mucosal biopsies are only slightly influenced by the efficacy of different microbiome analysis platforms. Furthermore, we investigated whether recent revisions to the International Code of Nomenclature for Prokaryotes and consequent alterations in taxonomic classification influenced the findings.
Gastric biopsy specimens were obtained from gastric cancer patients with well-defined clinical profiles (n=40), including individuals with and without Helicobacter pylori (H. pylori) infection, and from control subjects (n=39) with and without H. pylori infection. DNA extraction and 16S rRNA gene sequencing (V1-V2) was performed at one site, and the resulting fastQ files were distributed to five expert teams for independent analysis. Three widely used bioinformatics pipelines for microbiome analysis, namely DADA2, MOTHUR and QIIME2, were used for the analysis.

Results

The generated fastQ files exhibited no platform-dependent clustering, indicating the comparability of the sequences. Microbial diversity was slightly reduced in both GC groups and significantly reduced in the H. pylori positive controls. This is due to an extreme increase in the relative abundance of H. pylori in this group. It also follows that the samples of the H. pylori positive control group have a very high similarity and form the clearest cluster in the corresponding analysis. Regardless of the protocol used, H. pylori status, microbial diversity and relative bacterial abundance were reproducible on all platforms. Although minor differences in performance were detected and assessed, similarity analysis and bacterial enrichment assessment also yielded consistent results. The introduction of the new taxonomy only had a major impact on the Proteobacteria phylum, as it originally also included H. pylori according to the old taxonomy, but this is now assigned to the new Campilobacterota phylum.

Conclusion

In summary, our findings demonstrate that disparate microbiome analysis methodologies employed by independent research teams can yield comparable results from identical datasets.

Introduction

By 2030, pancreatic ductal adenocarcinoma (PDAC) is predicted to become the second leading cause of cancer-related death. Due to its resistance to conventional therapies and heterogeneous mutational landscape, molecular stratification and the development of targeted therapeutic options are essential for improving survival of PDAC patients. Previous work from our group investigated loss-of-function mouse models for the ATM serine/threonine kinase causing homologous recombination deficiency (HRD) in PDAC. Specifically, ATM loss was reported to accelerate tumorigenesis, support metastatic burden, and coincide with oncogenic programs, including epithelial-mesenchymal transition (EMT). Interestingly, ATM-deficient tumors accumulated EZH2, suggesting that ATM status contributes to rewiring the epigenetic landscape of pancreatic tumors, subsequently supporting oncogenic features.

Aims & Methods

To investigate whether and how EZH2 impacts ATM-deficient PDAC pathogenesis, we generated conditional double ATM and EZH2 knockouts in Kras^G12D/+; Ptf1a^Cre/+ mice. In vitro approaches were conducted using primary cell lines isolated from murine endpoint tumors. Additionally, CRISPR/Cas9 strategy was implemented on ATM-deficient cell lines to decipher the ATM-EZH2 molecular axis.

Results

Double-null mice showed extended mouse survival and decreased undifferentiated malignant cell features. Strikingly, interrogation of publicly available human datasets (TCGA, PanCancer Atlas) revealed a significant extended progression-free survival of ATM^neg EZH2^neg PDAC patients compared to ATM^neg EZH2^pos PDAC cases. Furthermore, we identified the histone-methyltransferase EZH2 as a direct ATM substrate impacting the mesenchymal phenotype of ATM-depleted tumors. Interestingly, RNA-seq analysis of ATM-deficient pancreatic cancer cells revealed upregulation of a transcriptomic program associated with cell differentiation upon EZH2 depletion by CRISPR/Cas9 strategy. In line, EZH2 depletion restored differentiated tumor features ex vivo. Further in vitro investigations revealed that ATM status significantly impacts EZH2 activity via a post-translational regulation axis, leading to a fostered tumor supporting EZH2 activity during pancreatic carcinogenesis. Specifically, the ATM kinase activity results in EZH2 ubiquitination and degradation by promoting Cullin1 E3-ubiquitin ligase recruitment. Finally, we demonstrated that a combinatorial therapy involving a potent EZH2 inhibitor promotes genomic instability and exacerbates the cytotoxic effects of PARP interference in ATM-deficient HRD PDAC.

Conclusion

Overall, we show that ATM deficiency significantly impacts EZH2 epigenetic functions and highlight the molecular stratification according to ATM status prior to pharmacological EZH2 interference in PDAC therapies as a valuable approach to target ATM-deficient HRD tumors.