Introduction

Long non-coding RNAs (lncRNAs) are involved in disease pathogenesis, and the roles of lncRNAs during pancreatic carcinogenesis remain poorly understood. Some lncRNAs are found in extracellular vesicles (EVs); EV-encapsulated RNAs in serum (obtained via liquid biopsy) have been used as biomarkers. We previously verified that the lncRNA highly upregulated in liver cancer (HULC) promotes invasion and migration by regulating the epithelial-mesenchymal transition (EMT) via EV transfer in pancreatic ductal adenocarcinoma (PDAC) cells. However, little is known about whether “EV lncRNAs” could serve as biomarkers or regulators of tumor growth in PDAC.

Aims & Methods

We aimed to identify novel EV lncRNAs that serve as serum biomarkers for PDAC and to investigate the functional roles mediated by lncRNAs in PDAC development. EV RNA was isolated from the serum of 3 PDAC patients and 3 healthy individuals. Gene expression in the serum EVs was examined using the Affymetrix cDNA microarray. LncRNA expression levels and functions were assessed using human pancreatic ductal epithelial (HPDE) and PDAC (e.g., Panc-1) cells. siRNAs and lentiviral vectors were used to modulate RNA expression. Cell invasion, migration, and apoptosis were examined by transwell, wound healing, and caspase 3/7 assays. Expression of RNA was assessed by qPCR and of protein by Western blot. Serum samples were collected from patients with PDAC (n=22) and intraductal papillary mucinous neoplasms (IPMNs; n=23) and healthy individuals (n=21). EV lncRNA expression in serum was analyzed by digital PCR. The diagnostic accuracy of various markers was evaluated using receiver operating characteristic (ROC) analysis, with measurement of the area under the curves (AUC) values.

Results

Gene expression analyses detected 1,205 genes that exhibited two-fold differential expression; the levels of 1,069 increased, and those of 136 decreased, in serum EVs of PDAC patients compared to those of healthy individuals. Of these lncRNAs, LINC02280 and LOC100507412 (termed HEVEPA) were the most significantly upregulated; both were highly expressed in a panel of PDAC relative to HPDE cells. In Panc-1 cells, knockdown of LINC02280 by siRNA significantly increased expression of E-cadherin and decreased expression of N-cadherin, Snail, and Vimentin, and cell invasion and migration. Moreover, overexpression of LINC02280 significantly reduced the expression of E-cadherin and increased the expression of N-cadherin, Snail, and Vimentin, and cell invasion and migration. Altogether, LINC02280 promoted cell invasion and migration through the induction of EMT. Furthermore, we found that HEVEPA inhibited the apoptosis of Panc-1 cells. These observations suggested that LINC02280 and HEVEPA would be oncogenic. In serum EVs, the expression levels of LINC02280, HEVEPA, and HULC were significantly elevated in PDAC patients compared to healthy individuals and IPMN patients (p < 0.05). The AUC values of the combinations [LINC02280, HEVEPA, HULC, and CA19-9] and [LINC02280, HEVEPA, and HULC] were higher than those of LINC02280, HEVEPA, HULC, or CA19-9 alone (1.00 and 0.98 vs. 0.89, 0.80, 0.92, and 0.89, respectively) in terms of discriminating PDAC from healthy individuals and IPMN patients.

Conclusion

LINC02280 and HEVEPA promote tumor development in PDAC. A combination of EV lncRNAs and CA19-9 showed good performance in predicting PDAC diagnosis and will find applications in liquid biopsies to determine human PDAC status.

Introduction

In the context of pancreatic ductal adenocarcinoma (PDAC) ≤ 10 mm in diameter, the diagnostic performance of endoscopic ultrasound-guided fine needle aspiration/biopsy (EUS-FNAB) is relatively low. Pancreatic juice cytology (PJC) has recently gained attention because of its high sensitivity for small PDAC. However, the appropriateness of EUS-FNAB/PJC for small PDAC is still controversial.

Aims & Methods

This study aimed to clarify the true diagnostic ability of EUS-FNAB and the salvage ability of PJC in PDAC ≤ 10 mm. A retrospective analysis was conducted on 271 patients with pancreatic tumors ≤ 10 mm confirmed by EUS, who were undergoing attempted EUS-FNAB. For accurate evaluation of the ability of EUS-FNAB to distinguish PDAC, pancreatic metastasis/malignant lymphoma were excluded. The technical success of EUS-FNAB was defined as the possibility of needle puncture and the ability to obtain adequate specimens for cytological/histological assessment. The diagnostic ability of EUS-FNAB was analyzed using technical success cases. A positive cytological diagnosis was defined as cells with highly suspected adenocarcinoma/definite adenocarcinoma based on the Bethesda system in both EUS-FNAB and PJC examinations. EUS-FNAB histological diagnosis was considered negative if only atypical cells existed. The diagnoses made with EUS-FNAB were determined based on cytology and/or histological findings. After eliminating technical failure or negative results by EUS-FNAB, patients with a strong likelihood of having PDAC from imaging characteristics underwent salvage PJC. The primary endpoint was clarifying the technical success rate/diagnostic ability of EUS-FNAB for PDAC ≤ 10 mm. In addition, the salvage ability of PJC for false-negative/technical failure EUS-FNAB cases was also examined.

Results

EUS-FNAB procedures were attempted for 271 patients with pancreatic tumors ≤ 10 mm. Overall, 80.8% of patients with pancreatic tumors (median lesion size of 8 mm [range, 3–10 mm]) achieved technical success. The reasons for technical failure were the inability to puncture due to anatomical inaccessibility (3.3%), unclear visibility for a puncture (5.2%), and inadequate specimen retrieval for cyto-histological analysis (10.7%).Actual diagnostic ability of EUS-FNAB in the diagnosis of PDAC ≤ 10 mm. In patients who achieved EUS-FNAB technical success, PDAC ≤ 10 mm was detected in 28.3% (62/219) of the patients with surgical resection/clinical follow-up. The sensitivity, specificity, and accuracy of comprehensive EUS-FNAB was 82.3%, 94.9%, and 91.3%, respectively. In the diagnostic ability comparison using AUC, comprehensive EUS-FNAB had naturally higher diagnostic ability than EUS-FNAB cytology alone/EUS-FNAB histology alone (P = 0.002, P < 0.001, respectively). In addition, EUS-FNAB cytology had higher diagnostic capability than EUS-FNAB histology alone (P = 0.034). Thirty-five patients were diagnosed with PDAC ≤ 10 mm from among the 212 patients who encountered technical failure/negative diagnosis by EUS-FNAB. Among these patients with PDAC ≤ 10 mm, 74.3% were additionally diagnosed by salvage PJC accurately (PJC positive and final diagnosis of PDAC ≤ 10 mm). The overall accuracy of PJC was 81.6% in this study. The other nine patients with PDAC ≤ 10 mm, who had not been accurately diagnosed by EUS-FNAB/PJC, were finally diagnosed by surgical resection/clinical follow-up based on their imaging findings.

Conclusion

The true technical success rate/sensitivity of EUS-FNAB for PDAC ≤ 10 mm was relatively low. EUS-FNAB followed by PJC may be ideal for diagnosing PDACs ≤ 10 mm in diameter.

Disclosure

Conflicts of interest: None declared.

Introduction

New-onset diabetes (NOD) is associated with the development of pancreatic ductal adenocarcinoma (PDAC) and is suggested as potential biomarker to detect PDAC at an earlier stage. International guidelines recommend routine glucose measurements in pancreatic surveillance of high-risk individuals (HRIs), however, little is known about its added value in addition to imaging. Therefore, this study evaluates the added value of longitudinal glucose measurements as a diagnostic biomarker for PDAC in high-risk surveillance cohorts.

Aims & Methods

HRIs with a CDKN2A germline pathogenic variant participating in pancreatic cancer surveillance who had ≥ 1 fasting blood glucose (FBG) and/or HbA1c measurements were included in this study. Pancreatic surveillance consists of annual magnetic resonance imaging (MRI) and endoscopic ultrasound (EUS), and blood sampling including blood glucose measurements. Individuals with diabetes mellitus at baseline, less than 3 years of follow-up and all follow-up after pancreatectomy were excluded from the study. Data was collected on demographics, FBG- and HbA1c measurements, MRI- and EUS examinations, and pathology reports. NOD was defined as two consecutive FBG values of ≥ 7 mmol/L or one HbA1c value of ≥ 48 mmol/mol. Univariate multivariable logistic regression was performed to assess the relationship between NOD and PDAC, with adjustment for sex, age and smoking history. To quantify the diagnostic performance of NOD as a marker for PDAC, receiver operating characteristic (ROC) curve and area under the curve (AUC) were computed.

Results

In total, 220 HRIs were included in the analysis. Median age was 61 (IQR 53-71) years, 62.7% of participants were female, and the mean BMI was 26.2 (4.12 SD). More than half (54.1%) of the studied cohort had a positive smoking history and the median amount of glucose measurements per person was 7 (IQR 5-12). During the study period, 26 (11.8%) HRIs developed NOD, of whom 5 (19.2%) later developed PDAC. The other 23 (82.1%) PDAC cases remained NOD-free. Multivariable analysis showed no statistically significant relationship between NOD and PDAC (OR 1.21; 95% CI, 0.39-3.78). A statistically significant association was exclusively observed between age and development of PDAC (OR 1.05; 95% CI, 1.01-1.1). Furthermore, NOD did not differentiate between HRIs with- and without PDAC (AUC 0.54; 95% CI, 0.46-0.61).

Conclusion

This study demonstrated no added value for longitudinal glucose monitoring in HRI participating in an imaging-based pancreatic cancer surveillance program.

Introduction

Pancreatic cancer surveillance in high-risk individuals has demonstrated improved survival outcomes resulting from diagnosis of early-stage PDAC and increased surgical resection rates. However, pancreatic surgery is associated with significant postoperative morbidity. This study compared short-term surgical outcomes in patients with a germline CDKN2A pathogenic variant (PV) under pancreatic cancer surveillance with symptomatic patients undergoing surgical resection for PDAC.

Aims & Methods

This was a single-center cohort study of prospectively collected data from the pancreatic cancer surveillance program and Dutch Pancreatic Cancer Audit (DPCA) of the Leiden University Medical Center (LUMC), a high-volume center for pancreatic cancer surgery. We included all patients with a CDKN2A PV under pancreatic cancer surveillance who underwent surgical resection for a suspected PDAC at the LUMC between 2000 and 2022 (i.e. pancreatoduodenectomy, distal pancreatectomy, and total pancreatectomy). Short-term surgical outcomes were compared to a control group of symptomatic patients with sporadic PDAC undergoing surgical resection at the same center between 2013 and 2021 using logistic regression analysis. The primary endpoint was major complications (Clavien-Dindo ≥ III) within 30 days after surgery. Secondary endpoints included all pancreatic surgery-specific complications, in-hospital mortality, reinterventions, length of hospital stay, and readmission within 30 days.

Results

In total, 32 patients in the surveillance group underwent surgical resection for suspected PDAC. Histopathology revealed benign lesions in 8 patients (25%) and malignancy in 24 patients (75%), including 23 PDAC and 1 ampullary carcinoma. A total of 247 patients in the control group with sporadic PDAC underwent surgical resection. In the surveillance group, patients were younger (59 [SD ±9] years vs. 67 [SD ±9] years, respectively) and had lower American Society of Anesthesiologists (ASA) scores than the sporadic PDAC group (ASA ≥ III, 0% vs. 30%; Table 1). In addition, tumors diagnosed under surveillance were smaller (15 mm [IQR 6-30] vs. 28 mm [IQR 20-35]) and more often located in the pancreatic body or tail compared with sporadic PDACs (53% vs. 17%). Consequently, distal pancreatectomy was performed more frequently in the surveillance group than in the sporadic PDAC group (50% vs. 14%). After surgical resection, no difference in major complications was found between the surveillance group and the sporadic PDAC group (25% vs. 20%, P=0.50). However, the surveillance group developed more postoperative pancreatic fistulas than the sporadic PDAC group (22% vs. 6%, P<0.01). Mortality, reinterventions, length of hospital stay, and readmission within 30 days did not differ between groups.

Table 1. Patient, tumor, and treatment characteristics

Conclusion

Major complications did not differ between the surveillance and sporadic PDAC groups, despite differences in patient and tumor characteristics including age, ASA score, tumor size, and location. However, patients in the surveillance group had a higher risk of developing postoperative pancreatic fistula.

Introduction

Metabolic dysfunction-associated steatotic liver disease (MASLD), formerly known as nonalcoholic fatty liver disease (NAFLD), is a widespread chronic liver condition closely associated with obesity, insulin resistance, and dyslipidemia. Recent evidence points to gut microbiota dysbiosis as a key player in MASLD pathogenesis, affecting intestinal barrier function and systemic inflammation.

Aims & Methods

The aim was to investigate gut microbiota composition in patients with MASLD and assess its association with disease progression and gut barrier dysfunction. A total of 152 MASLD patients and 150 matched controls without liver pathology were examined. Diagnosis was confirmed by ultrasound and steatometry, alongside assessment of cardiometabolic risk factors. Gut microbiota composition was analyzed using real-time PCR, focusing on Bacteroidetes, Firmicutes, and Actinobacteria. Hydrogen breath testing was used to diagnose small intestinal bacterial overgrowth (SIBO), and the presence of Akkermansia muciniphila was evaluated as a marker of gut barrier integrity.

Results

MASLD patients demonstrated a significant increase in Firmicutes (50.3%) and Actinobacteria (15.3%) proportions compared to controls, with a higher Firmicutes/Bacteroidetes (F/B) ratio. SIBO was detected in 52% of MASLD patients versus 40.5% in controls (p<0.05). The presence of Akkermansia muciniphila was notably lower in MASLD patients (33%) compared to controls (63%), suggesting impaired gut barrier function. Strong positive correlations were observed between Actinobacteria abundance and the F/B index (r=0.75, p<0.05), while Akkermansia muciniphila presence negatively correlated with MASLD development (r=–0.69, p<0.05).

Conclusion

Gut microbiota dysbiosis, characterized by elevated Firmicutes and Actinobacteria and reduced Akkermansia muciniphila, is closely associated with MASLD pathogenesis. The findings highlight the potential of microbiota-targeted interventions for improving gut barrier function and modifying disease progression in MASLD patients.