Introduction

Chemotherapeutical treatment of pancreatic cancer guided by drug screening of patient-derived tumor organoids (PDTO) is a promising new tool in personalized medicine. However, standard PDTO models do not adequately model the tumor microenvironment, which have been recognized to play a pivotal role in tumor progression, maintenance and therapeutic response. Cancer-associated fibroblasts are dominant mediators of these effects and co-cultures including both PDTOs and CAFs from surgical specimens show that addition CAFs modify resistance of cancer cells to common chemotherapeutic drugs in vitro. However, as previous co-culture models have been established from surgical specimens, they cannot be applied to patients with inoperable disease. To address these shortcomings, we aimed to create a PDTO/CAF co-culture model derived from endoscopic ultrasound-guided fine-needle biopsies (EUS-FNBs) which could be readily used for drug screening purposes.

Aims & Methods

PDTO and CAF cultures were isolated and expanded from EUS-FNBs taken during diagnostic procedures in patients undergoing diagnostic workup for PDAC. Co-cultures were created after thorough characterization and validation of PDTOs and CAFs using next generation sequencing, immunohistochemistry and histopathological evaluation. Interaction between cell types in our co-culture model were evaluated by measuring area of PDTOs in co-cultures vs. standard PDTO cultures.

Results

Validated PDTO and CAF cultures could be expanded from EUS-FNBs with a success rate of 19% and 25%, respectively. In total, eight co-cultures from five different patients were created. A significant increase in growth of PDTOs was observed in 50% of the co-cultures compared to monocultures.

Conclusion

Advanced organoid co-culture models which are able to capture CAF/PDTO interaction can be established from EUS-FNB samples. However, clinical application is hampered by low success rates

References

[1] Tiriac H, Belleau P, Engle DD, et al. Organoid Profiling Identifies Common Responders to Chemotherapy in Pancreatic Cancer. Cancer Discov. 2018;8(9):1112-1129. doi:10.1158/2159-8290.CD-18-0349
[2] Beutel AK, Schütte L, Scheible J, et al. A Prospective Feasibility Trial to Challenge Patient–Derived Pancreatic Cancer Organoids in Predicting Treatment Response. Cancers (Basel). 2021;13(11):2539. doi:10.3390/cancers13112539
[3] Kalluri R. The biology and function of fibroblasts in cancer. Nat Rev Cancer. 2016;16(9):582-598. doi:10.1038/nrc.2016.73
[4] Schuth S, Le Blanc S, Krieger TG, et al. Patient-specific modeling of stroma-mediated chemoresistance of pancreatic cancer using a three-dimensional organoid-fibroblast co-culture system. J Exp Clin Cancer Res. 2022;41(1):312. doi:10.1186/s13046-022-02519-7

Introduction

Blood-based liquid biopsy for comprehensive genomic profiling is available for cancer patients; however, given the limited amount of circulating tumor-derived DNA in plasma, it remained challenging to detect pancreatic ductal adenocarcinoma (PDA) at earlier stages. We sought to determine the usefulness of pancreatic juice-containing DNA collected from a site more proximal to the tumor in the early diagnosis of pancreatic cancer.

Aims & Methods

We evaluated the mutation detection in DNA from the duodenal fluid as an alternative biospecimen obtained by a less-invasive approach relative to endoscopic biopsy. In 296 patients diagnosed with PDA (n=175), IPMN (n=61), or other pancreatic diseases (n=60) were recruited in seven institutions. Duodenal fluid was collected from the duodenal lumen through the endoscopic channel and pancreatic juice was obtained directly from the pancreatic duct through a catheter during the ERP examination. Together with plasma cfDNA, both digestive fluid-included DNA was extracted, and molecular barcode sequencing (Ion Amplseq HD) targeting five pancreatic cancer-associated genes, namely, KRAS, GNAS, TP53, CDKN2A, and SMAD4, were performed. Detected mutations were evaluated for concordance with surgically resected tissue or needle biopsy.

Results

DNA extracted from the duodenal fluids (DFDNA) and pancreatic juice (PjDNA) had a much higher yield (avg. 25.4 ng/mL, and 7.7 ng/mL, respectively) than plasma cell-free DNA (avg. 1.3 ng/ml). While most plasma cfDNA had a typical fragmentation pattern with the highest peak at 160 bp, DFDNA and PjDNA showed various fragmentation patterns. Using molecular barcode sequencing, hotspot mutations detected in the DFDNA (n=148) showed 50.7% concordance of the mutations in resected PDA lesions, whereas only 24.1% concordance in the plasma cfDNA (n=114). In contrast to plasma cfDNA, where liquid-tissue matches were limited to advanced stages of cancer, DFDNA assay enabled the higher concordance through a wide range of PDA stages, even Stage 0 (83.3%) or I (45.5%) UICC 8th, or IPMN cases. Furthermore, PjDNA showed a much higher concordance rate (88%, n=25) than DFDNA. In DFDNA and PjDNA, mutations that originated from the primary lesions and from multicentric/concomitant lesions such as IPMN were detected effectively.

Conclusion

Digestive fluid-derived DNA can be a potent biospecimen to detect mutations with higher yield and sensitivity than plasma cfDNA. For practical diagnostics testing, optimized collection and storage of the duodenal fluid and methods for purification of the nucleic acids are needed. Besides as a diagnostic tool, genomic profiling of DFDNA can offer better surveillance for individuals at high-risk or annual checkups for healthy people. PjDNA can predict the presence of neoplastic lesions with extremely high probability and has a powerful potential for further testing before a more definitive diagnosis, even without histological evidence of cancer.

Disclosure

Yusuke Ono, Kenji Takahashi, Yusuke Mizukami: Research grants from Hitachi High-Tech Corporation.

Introduction

PNET is an important determinant of survival in MEN1 patients and the most important cause of MEN1-related death. Early identification of pNETs in MEN1 is important since these represent a common cause of morbidity and mortality of the syndrome. Prognosis is associated with tumour size. Many pNETs are small subcentimetre lesions and only become symptomatic when the tumour is large. Conventional radiology is usually suboptimal in detecting small tumours (< 2cm), thus missing the window to remove them at an earlier stage. We aim to compare EUS detection of pNETs in patients with confirmed MEN1 compared to cross-sectional imaging including CT and/or MRI pancreas and assess the incremental benefit of EUS.

Aims & Methods

Over a span of 7 years, a total of 20 patients with clinically confirmed MEN1 had surveillance of pNET performed with EUS, CT/MRI and biochemical screening. Data were retrospectively retrieved from the hospital electronic records database. EUS and CT/MRI done within 3-6 months of each other were compared to minimize discrepancy in findings due to pNET size progression and new lesions over time. Statistical analysis was performed using SPSS v20 on the size and number of pNET detection by EUS and CT/MRI using the Wilcoxon Signed Rank Test and McNemar Chi-square.

Results

A total of 28 EUS procedures and an equal number of cross sectional imaging (CT/MRI) were performed. pNETs were identified in 95% of all 20 MEN1 cases. Overall median pNET size was 7.1mm on EUS and 14.5mm on CT/MRI (p=0.007). Mean value for smallest PNETs detected by EUS was 4.6mm and 12.7mm on CT/MRI (p=0.001). EUS detected more pancreatic lesions/pNETs compared to CT/MRI (p<0.001) in 25 of the 28 procedures (89.3% more). The remaining 3 procedures showed equal numbers of pNETs detection by both modalities. The interquartile range (IQR) for smallest pNET detected by EUS was 3.0-5.0mm while IQR for CT/MRI lies between 8.3mm-14.8mm. EUS detected all 100% cases of pNETs in our series of MEN1 compared to CT/MRI imaging alone which detected 57.9% cases (p=0.008). 14 of 20 patients had FNA performed with a positive yield of 85.7%. In 50% of patients, pNET measured ≤10 mm. 45% of patients underwent surgery for pNETs while the rest had surveillance.

Conclusion

In MEN1 patients, CT/MRI underestimated the presence of pancreatic lesions/pNETs in 42.1% compared to EUS (p=0.008) and was not able to identify pNETs < 8mm in all but one case.
EUS offers higher sensitivity than cross sectional imaging (CT/MRI) in terms of detecting the number of positive pNET cases as well as a greater number of pancreatic lesions especially subcentimetre lesions in MEN1. EUS should be considered a standard tool in the algorithm for MEN1 workup, instead of an adjunct reserved for diagnostic dilemmas.

References

NCCN Guidelines 2015 version 1.2015 Neuroendocrine Tumours.

Introduction

Chronic pancreatitis (CP) is a complex inflammatory disorder characterized by progressive destruction of the pancreatic tissue, with fibrotic transformation of the tissue as a major morphological feature. The incidence of the disease is steadily increasing, but no specific therapy is currently available, making the identification of new therapeutic targets a priority. Plasminogen activator inhibitor-1 (SERPINE1/PAI-1) has been implicated in various pathological processes, including fibrosis and inflammation. However, its specific involvement in the pathogenesis of CP is not yet fully understood.

Aims & Methods

We therefore aimed to explore the role of PAI-1 in the development of CP and assess the therapeutic potential of PAI-1 inhibitors as antifibrotic agents to prevent the progression of CP.
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​The PAI-1 expression of the pancreatic tissue was determined in mouse and human pancreatic tissue samples with immunohistochemistry. CP was induced in FVB/N mice with 8X50 µg/kgbw i.p. cerulein injection, given every hour, every 3 days for a total of 5 times. PAI-1 activity was inhibited with a specific inhibitor, which was administered per os at a dose of 10 mg/kgbw for 6 consecutive days after the 3rd cerulein treatment. To assess the extent of inflammation and fibrosis hematoxylin-eosin and Crossman’s trichrome staining were performed, and biochemical assays were used to determine amylase activity and hydroxyproline concentration. PAI-1 expression and location in the pancreas was examined by RT-qPCR and immunofluorescence co-staining.

Results

PAI-1 expression was significantly higher in pancreatic tissue samples obtained from CP patients. After induction of CP in mice, the body weight and the pancreatic weight/body weight ratio significantly reduced, whereas histopathology showed significant pancreatic atrophy and fibrosis. This was also confirmed by the increased hydroxyproline concentration and decreased amylase activity of the pancreatic tissue. The gene expression of SERPINE1 increased in pancreas tissue and fibroblast cell culture in CP group, moreover the elevated PAI-1 signal localization suggests a possible expression role for activated stellate cells. Administration of PAI-1 inhibitor resulted in a decrease in both body weight and pancreatic weight loss rates, additionally the atrophy, tissue fibrosis, hydroxyproline concentrations and SEPRINE1 gene expression were significantly decreased compared to the CP group.

Conclusion

Our results indicate an elevation in PAI-1 expression in CP, with the potential for fibrosis and tissue atrophy reduction by the inhibition of PAI-1. These findings propose PAI-1 as a promising therapeutic target for the treatment of chronic pancreatitis.