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A 55-yr-old woman presented with abdominal bloating for approximately 1 year. Imaging studies showed a cyst in the body of the pancreas with proximal pancreatic ductal dilation. An endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) was performed. Cytologic findings from the cyst fluid were consistent with a mucinous neoplastic cyst, and the possibility of malignancy could not be entirely excluded. A KRAS mutation analysis was performed on the cyst fluid using the Idylla system and circulating tumor KRAS (ctKRAS) cartridge (Biocartis, Mechelen, Belgium), which tests for actionable mutations in exons 2, 3, and 4 of the KRAS gene. Idylla testing detected a KRAS G12D mutation in the cyst fluid. The patient subsequently underwent a distal subtotal pancreatectomy with splenectomy. Microscopic examination of the resected tissue revealed an intraductal papillary mucinous neoplasm (IPMN) with an associated invasive carcinoma. KRAS testing on the resected tumor tissue confirmed the G12D mutation detected in the cyst fluid earlier. The described rapid testing of KRAS directly from the pancreatic cyst fluid can complement cytology assessment to classify pancreatic cysts more reliably and can potentially be of significant help when other cyst findings are nondiagnostic.
It is clear that specific intestinal bacteria are involved in the development of different premalignant conditions along the gastrointestinal tract. An analysis of the microbial constituents in the context of pancreatic cystic lesions has, however, as yet not been performed. This consideration prompted us to explore whether endoscopically obtained pancreatic cyst fluids (PCF) contain bacterial DNA and to determine the genera of bacteria present in such material.
Pancreatic cyst fluids (PCFs) enriched in tumour-derived proteins are considered a potential source of new biomarkers. This study aimed to determine compositional and quantitative differences between the degradome and proteome of PCFs aspirated from different types of pancreatic cyst lesions (PCLs). 91 patients who underwent endoscopic ultrasound-fine needle aspiration under routine clinical diagnosis of PCLs were enrolled. Four cysts were malignant (CAs), and 87 were nonmalignant and consisted of 18 intraductal papillary mucinous neoplasms (IPMNs), 14 mucinous cystic neoplasms (MCNs), nine serous cystic neoplasms (SCNs), 29 pseudocysts (PCs), and 17 unclassified. Profiles of the <5 kDa fraction, the degradome, and the trypsin-digested proteome were analysed using an LTQ-Orbitrap Elite mass spectrometer coupled with a nanoACQUITY LC system. Qualitative analyses identified 796 and 366 proteins in degradome and proteome, respectively, and 689 (77%) and 285 (78%) of them were present in the Plasma Proteome Database. Gene Ontology analysis showed a significant overrepresentation of peptidases and peptidases inhibitors in both datasets. In the degradome fraction, quantitative values were obtained for 6996 peptides originating from 657 proteins. Of these, 2287 peptides were unique to a single type, and 515 peptides, derived from 126 proteins, were shared across cyst types. 32 peptides originating from 12 proteins had differential (adjusted p-value ≤0.05, FC ≥1.5) abundance in at least one of the five cysts types. In proteome, relative expression was measured for 330 proteins. Of them, 33 proteins had significantly (adjusted p-value ≤0.05, FC ≥1.5) altered abundance in at least one of the studied groups and 19 proteins appeared to be unique to a given cyst type. PCFs are dominated by blood proteins and proteolytic enzymes. Although differences in PCF peptide composition and abundance could aid classification of PCLs, the unpredictable inherent PCF proteolytic activity may limit the practical applications of PCF protein profiling.
Pancreatic cystic neoplasms (PCNs) are recognized as precursor lesions of pancreatic cancer, with a marked increase in prevalence. Early detection of malignant PCNs is crucial for improving prognosis; however, current diagnostic methods are insufficient for accurately identifying malignant PCNs. Here, we utilized mass spectrometry (MS)-based glycosite- and glycoform-specific glycoproteomics, combined with proteomics, to explore potential cyst fluid diagnostic biomarkers for PCN. The glycoproteomic and proteomic landscape of pancreatic cyst fluid samples from PCN patients was comprehensively investigated, and its characteristics during the malignant transformation of PCN were analyzed. Under the criteria of screening specific cyst fluid biomarkers for the diagnosis of PCN, a group of cyst fluid glycoprotein biomarkers was identified. Through parallel reaction monitoring (PRM)-based targeted glycoproteomic analysis, we validated these chosen glycoprotein biomarkers in a second cohort, ultimately confirming N-glycosylated PHKB (Asn-935, H5N2F0S0; Asn-935, H4N4F0S0; Asn-935, H5N4F0S0), CEACAM5 (Asn-197, H5N4F0S0) and ATP6V0A4 (Asn-367, H6N4F0S0) as promising diagnostic biomarkers for distinguishing malignant PCNs. These glycoprotein biomarkers exhibited robust performance, with an area under the curve ranging from 0.771 to 0.948. In conclusion, we successfully established and conducted MS-based glycoproteomic analysis to identify novel cyst fluid glycoprotein biomarkers for PCN. These findings hold significant clinical implications, providing valuable insights for PCN decision-making, and potentially offering therapeutic targets for PCN treatment.
DNA mutational analysis of pancreatic cystic fluid (CF) is a useful adjunct to the evaluation of pancreatic cysts. KRAS/GNAS or RAF/PTPRD/CTNNB1/RNF43 mutations are highly specific to precancerous or advanced neoplasia. Several studies recently demonstrated the ability of next-generation sequencing (NGS) analysis to detect DNA mutations in pancreatic CF, but few studies have performed a systematic comparative analysis between pancreatic CF and neoplastic surgical tissue (NT). The value of CF-NGS analysis indicators for determining surgical resection necessitates evaluation.
DNA-based testing of pancreatic cyst fluid (PCF) is a useful adjunct to the evaluation of pancreatic cysts (PCs). Mutations in KRAS/GNAS are highly specific for intraductal papillary mucinous neoplasms (IPMNs) and mucinous cystic neoplasms (MCNs), while TP53/PIK3CA/PTEN alterations are associated with advanced neoplasia. A prospective study was performed to evaluate preoperative PCF DNA testing.
Current efforts in the identification of new biomarkers are directed towards an accurate differentiation between benign and premalignant cysts. Thermal Liquid Biopsy (TLB) has been previously applied to inflammatory and tumor diseases and could offer an interesting point of view in this type of pathology.
Next-generation sequencing (NGS) of pancreatic cyst fluid is a useful adjunct in the assessment of patients with pancreatic cyst. However, previous studies have been retrospective or single institutional experiences. The aim of this study was to prospectively evaluate NGS on a multi-institutional cohort of patients with pancreatic cyst in real time.
The application of advanced imaging technologies for identifying pancreatic cysts has become widespread. However, accurately differentiating between low-grade dysplasia (LGD), high-grade dysplasia (HGD), and invasive intraductal papillary mucinous neoplasms (IPMNs) remains a diagnostic challenge with current biomarkers, necessitating the development of novel biomarkers that can distinguish IPMN malignancy.
Pancreatic cystic lesions (PCL) are increasingly diagnosed. Endoscopic ultrasound fine-needle aspiration (EUS-FNA) cytology is often used for diagnostic confirmation but can be inconclusive. In this study, the role of molecular analyses in the pre-operative diagnostics of PCL is evaluated. Targeted Next Generation Sequencing (NGS) applied on cytology smears was retrospectively evaluated in a cohort of 37 resected PCL. Usefulness of NGS on fresh cyst fluids was tested in a prospective cohort of patients with newly diagnosed PCL (n = 71). In the retrospective cohort, cytology plus NGS displayed higher sensitivity (94.1% vs. 87.1%) and specificity (100% vs. 50%) than cytology alone for the detection of mucinous neoplasms. In the prospective cohort, sensitivity and specificity of conventional cytology alone were 54.2% and 100% for the detection of mucinous neoplasia and 50.0% and 100% for the detection of high-grade dysplasia, respectively. Adding NGS, all lesions which underwent histopathologic verification (12/71, 17%) could be classified without false positive or false negative results regarding the detection of mucinous neoplasm so far. NGS analysis of cfDNA in PCL fluids is feasible and can increase diagnostic accuracy in the detection of mucinous neoplasms compared to cytology alone. However, algorithms for the detection of high-risk lesions need further improvement.
Despite the increasing rate of detection of incidental pancreatic cystic lesions (PCLs), current standard-of-care methods for their diagnosis and risk stratification remain inadequate. Intraductal papillary mucinous neoplasms (IPMNs) are the most prevalent PCLs. The existing modalities, including endoscopic ultrasound and cyst fluid analysis, only achieve accuracy rates of 65-75% in identifying carcinoma or high-grade dysplasia in IPMNs. Furthermore, surgical resection of PCLs reveals that up to half exhibit only low-grade dysplastic changes or benign neoplasms. To reduce unnecessary and high-risk pancreatic surgeries, more precise diagnostic techniques are necessary. A promising approach involves integrating existing data, such as clinical features, cyst morphology, and data from cyst fluid analysis, with confocal endomicroscopy and radiomics to enhance the prediction of advanced neoplasms in PCLs. Artificial intelligence and machine learning modalities can play a crucial role in achieving this goal. In this review, we explore current and future techniques to leverage these advanced technologies to improve diagnostic accuracy in the context of PCLs.
Perturbing redox homeostasis potentially constitutes a selective cancer-killing strategy. An engineered human enzyme, cyst(e)inase that degrades extracellular cysteine (l-Cys) and cystine (CSSC) leading to depletion of intracellular l-Cys and glutathione (GSH) was evaluated for its effects on pancreatic cancer cell lines. Cyst(e)inase caused oxidative stress and apoptosis in only Panc1 cells, whereas MIA-PaCa2 and BxPC3 cells demonstrated survival under conditions of cyst(e)inase-mediated l-Cys depletion through maintenance of mitochondrial metabolism and lower levels of reactive oxygen species (ROS). A correlation was also observed between thioredoxin 1 protein levels and resistance to cyst(e)inase treatment. Notably, cyst(e)inase in combination with auranofin, a thioredoxin reductase inhibitor, caused a synergistic increase in mitochondrial ROS and apoptosis and inhibition of mitophagy in the more resistant cells. In addition, auranofin treatment sensitized the more resistant pancreatic cancer xenografts to cyst(e)inase without systemic toxicity. These data provide strong rationale to further investigate therapeutic strategies that target multiple antioxidant pathways for treatment of pancreatic ductal adenocarcinoma.
The incidence of patients with pancreatic cystic lesions, particularly intraductal papillary mucinous neoplasm (IPMN), is increasing. Current guidelines, which primarily consider radiological features and laboratory data, have had limited success in predicting malignant IPMN. The lack of a definitive diagnostic method has led to low-risk IPMN patients undergoing unnecessary surgeries. To address this issue, we discovered IPMN marker candidates by analyzing pancreatic cystic fluid by mass spectrometry. A total of 30 cyst fluid samples, comprising IPMN dysplasia and other cystic lesions, were evaluated. Mucus was removed by brief sonication, and the resulting supernatant was subjected to filter-aided sample preparation and high-pH peptide fractionation. Subsequently, the samples were analyzed by LC-MS/MS. Using several bioinformatics tools, such as gene ontology and ingenuity pathway analysis, we detailed IPMNs at the molecular level. Among the 5834 proteins identified in our dataset, 364 proteins were differentially expressed between IPMN dysplasia. The 19 final candidates consistently increased or decreased with greater IPMN malignancy. CD55 was validated in an independent cohort by ELISA, Western blot, and IHC, and the results were consistent with the MS data. In summary, we have determined the characteristics of pancreatic cyst fluid proteins and discovered potential biomarkers for IPMN dysplasia.
Diagnosis of pancreatic cystic neoplasms remains problematic. We hypothesize that inflammatory mediator proteins in pancreatic cyst fluid can differentiate branch duct intraductal papillary mucinous neoplasms (BD-IPMNs) and pancreatic inflammatory cysts. We aim to 1) detect inflammatory mediator proteins (IMPs) using a multiplexed IMP-targeted microarray in pancreatic cyst fluid obtained during endoscopic ultrasound fine needle aspiration (EUS-FNA) and 2) compare IMP profiles in pancreatic cyst fluid from BD-IPMNs and inflammatory cysts. Pancreatic cyst fluid from ten patients (5 BD-IPMN and 5 inflammatory cysts) was obtained by EUS-FNA and analyzed directly with a multiplexed microarray assay to determine concentrations of 89 IMPs. Statistical analysis was performed using non-parametric methods.
Radioiodine (I-131) whole-body scintigraphy (WBS) is a useful modality for identifying functionally preserved thyroid tissue and metastases from differentiated thyroid cancer (DTC); however, the specificity of I-131 uptake is limited, and its accumulation in the pancreas has not been well described.
Diagnosis of malignant pancreatic cystic lesions (PCLs) is challenging as there is no investigation that offers both high diagnostic sensitivity and specificity for a definite diagnosis. Accurate diagnosis of cyst type is vital in order to not miss opportunities for early treatment of potentially malignant lesions and to avoid unnecessary surgeries. Serine protease inhibitor Kazal type I (SPINK1) and glucose are promising cyst fluid markers for differentiation of mucinous from non-mucinous cysts. We aim to validate the value of SPINK1 and glucose in detecting potentially malignant PCLs.
Although intraductal papillary mucinous neoplasm (IPMN) is thought to be a precursor lesion of pancreatic cancer, diagnosing malignant transformation of IPMN using non-invasive diagnostic methods is difficult and complicated. Micro-RNAs (miRNAs) are currently recognized as biomarkers and molecular targets of various diseases, including malignancy. In this study, we investigated a potential diagnostic approach using miRNA in pancreatic cyst fluid as a marker for evaluating malignant alternation of IPMN. Cystic fluid was sampled mainly during surgical resection. The collected samples were evaluated by performing comprehensive analysis of miRNA using a highly sensitive DNA chip. miRNA expression was compared between IPM adenoma (IPMA) and IPM carcinoma (IPMC) to evaluate the related biomarkers for malignant transformation of IPMN. miRNA analysis revealed that six miRNAs (miR-711, miR-3679-5p, miR-6126, miR-6780b-5p, miR-6798-5p, and miR-6879-5p) in IPMC were significantly enriched compared to those in IPMA. The difference was validated using quantitative real-time PCR. Cyst fluid miRNA analysis might be useful for diagnosing malignant alteration of IPMN. Further evaluations of diagnostic capability as well as functional analysis using the identified miRNAs are required with larger cohorts to confirm its efficacy.
The role of cyst fluid carcinoembryonic antigen (CEA) level in differentiating mucinous pancreatic cystic lesions (PCLs) is controversial. We investigated the role of cyst fluid CEA in differentiating low-risk (LR)-intraductal papillary mucinous neoplasms (IPMNs) from high-risk (HR)-IPMNs and LR-mucinous cystic neoplasms (MCNs).
Intraductal papillary mucinous neoplasm of the bile ducts (IPMN-B) is a true pre-cancerous lesion, which shares common features with pancreatic IPMN (IPMN-P). While IPMN-P is a well described entity for which guidelines were formulated and revised, IPMN-B is a poorly described entity.We carried out a systematic review to evaluate the existing literature, emphasizing the role of MRI in IPMN-B depiction.
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