Simple mucinous cyst: another potential cancer precursor in the pancreas? Case report with molecular characterization and systematic review of the literature.

Cystic lesions of the pancreas may range from benign to precursors of pancreatic cancer. Simple mucinous cyst (SMC) is larger than 1 cm, has a gastric-type flat mucinous lining, and minimal atypia without ovarian-type stroma. We report a new case of pancreatic SMC, coupling a systematic review of the English literature mainly focused on their clinic-pathological features. We reviewed 103 cases of SMC in adults (73 women), averaging 57 (range, 26-70) years. The SMCs were located in the body-tail region of the pancreas in 60 (58%) cases, presenting as single cystic lesions in 94% of cases; 43% of patients were asymptomatic. A preoperative fine-needle aspiration of the cyst fluid detected amylase and carcinoembryonic antigen positivity in 71% and 76% of cases, respectively. Patients underwent surgery mostly for suspected malignancy; in 83% of cases, a standard pancreatic resection was performed. Mean SMC size was 4.9 (range, 1.5-12.0) cm. Mucins MUC5AC and MUC6 resulted positive in 77% and 81% of cases performed, respectively, whereas MUC2 was negative in all but one patient. The SMC from our institution was characterized by a KRAS somatic mutation. The diagnosis of SMC should be considered when a solitary pancreatic cyst larger than 1 cm is detected in asymptomatic patients. To establish a correct diagnosis, an extensive histologic/immunohistochemical analysis is essential. The presence of a KRAS mutation highlights that SMC may represent another potential pancreatic cancer precursor.

Endoscopic Ultrasound-Guided Fine-Needle Aspiration Microhistology in Asymptomatic and Symptomatic Pancreatic Cystic Lesions.

OBJECTIVE: This study aimed to analyze the usefulness of endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) microhistology to detect malignancy in pancreatic cystic lesions (PCLs). METHODS: Patients with PCLs were identified and submitted to EUS-FNA from January 2010 to January 2017. The percentage of samples suitable for diagnostic classification by microhistology and the positive and negative likelihood ratios to detect malignancy in asymptomatic (APC) and symptomatic (SPC) PCLs were determined. RESULTS: Endoscopic ultrasound-guided fine-needle aspiration was performed in 510 patients. The resulting material was processed by microhistology and useful for diagnosis in 432 (84.2%). Clinical characteristics of APC (341) and SPC (169) revealed that APC patients were younger (P = 0.004) and had smaller PCLs (23 vs 35 mm; P < 0.001). In APC, we found more preneoplastic (38.7% vs 30.2%; P = 0.0016) and a lower number of malignant PCLs (8.2% vs 24.3%; P < 0.001). In APC and SPC, the sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of microhistology to detect malignancy were 71.4%, 99.7%, 95.2%, 97.5%, and 97.4% (k = 0.80) and 58.5%, 96.9%, 85.7%, 87.9%, and 87.6%, respectively. CONCLUSIONS: Endoscopic ultrasound-guided fine-needle aspiration was technically feasible. Microhistology was especially useful to detect neoplastic or malignant PCLs in APC patients.

Mutational Mosaics of Cell-Free DNA from Pancreatic Cyst Fluids.

BACKGROUND: Pancreatic cyst fluids (PCFs) enriched in tumor-derived DNA are a potential source of new biomarkers. The study aimed to analyze germinal variants and mutational profiles of cell-free (cf)DNA shed into the cavity of pancreatic cysts. METHODS: The study cohort consisted of 71 patients who underwent endoscopic ultrasound fine-needle aspiration of PCF. Five malignant cysts, 19 intraductal papillary mucinous neoplasms (IPMNs), 11 mucinous cystic neoplasms (MCNs), eight serous cystic neoplasms (SCNs), and 28 pseudocysts were identified. The sequencing of 409 genes included in Comprehensive Cancer Panel was performed using Ion Proton System. The mutation rate of the KRAS and GNAS canonical loci was additionally determined using digital PCR. RESULTS: The number of mutations detected with NGS varied from 0 to 22 per gene, and genes with the most mutations were: TP53, KRAS, PIK3CA, GNAS, ADGRA2, and APC. The frequencies of the majority of mutations did not differ between non-malignant cystic neoplasms and pseudocysts. NGS detected KRAS mutations in malignant cysts (60%), IPMNs (32%), MCNs (64%), SCNs (13%), and pseudocysts (14%), with GNAS mutations in 20%, 26%, 27%, 13%, and 21% of samples, respectively. Digital PCR-based testing increased KRAS (68%) and GNAS (52%) mutations detection level in IPMNs, but not other cyst types. CONCLUSIONS: We demonstrate relatively high rates of somatic mutations of cancer-related genes, including KRAS and GNAS, in cfDNA isolated from PCFs irrespectively of the pancreatic cyst type. Further studies on molecular mechanisms of pancreatic cysts malignant transformation in relation to their mutational profiles are required.

Cell block processing is optimal for assessing endoscopic ultrasound fine needle aspiration specimens of pancreatic mucinous cysts.

AIMS: The cell block technique for assessing endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) specimens from pancreatic mucinous cystic lesions (MCLs) was systematically evaluated for the first time, including comparisons with three traditional methods of assessing such specimens. METHODS: The prospective arm comprised EUS-FNA specimens from EUS-suspected pancreatic MCLs. The retrospective arm comprised EUS-FNA specimens from pancreatic MCLs surgically resected before the study start. For each specimen, these data points were collected: macroscopic likelihood of mucin, cyst fluid carcinoembryonic antigen (CEA) level and presence of mucin in air-dried, direct smears and in cell block preparations. RESULTS: The prospective and retrospective arms of the study comprised 80 and 30 EUS-FNA specimens, respectively. Seven prospective cases led to surgical resections during the study, and therefore, 37 EUS-FNA specimens were confirmed to have originated from MCLs. In the prospective arm, macroscopic mucin was suspected, cyst fluid CEA level exceeded 192 ng/mL, mucin was detected in direct smears and cell block preparations in 78%, 30%, 39% and 73% of cases, respectively. Of the 37 specimens confirmed to originate from MCLs, macroscopic mucin assessment, cyst fluid CEA level, direct smear mucin assessment and cell block mucin assessment had sensitivities for diagnosing MCL of 87%, 45%, 45% and 81%, respectively. CONCLUSIONS: Cell block preparations are as likely to identify mucin from pancreatic MCLs as macroscopic assessment but are twice as likely to diagnose MCL than direct smears and fluid CEA biochemistry. The cell block technique is easy for sample collection and processing especially because these are identical for solid and cystic pancreatic lesions.

Cross Validation of the Monoclonal Antibody Das-1 in Identification of High-Risk Mucinous Pancreatic Cystic Lesions.

BACKGROUND & AIMS: Although pancreatic cystic lesions (PCLs) are frequently and incidentally detected, it is a challenge to determine their risk of malignancy. In immunohistochemical and enzyme-linked immunosorbent assay (ELISA) analyses of tissue and cyst fluid from pancreatic intraductal papillary mucinous neoplasms, the monoclonal antibody Das-1 identifies those at risk for malignancy with high levels of specificity and sensitivity. We aimed to validate the ability of Das-1 to identify high-risk PCLs in comparison to clinical guidelines and clinical features, using samples from a multicenter cohort. METHODS: We obtained cyst fluid samples of 169 PCLs (90 intraductal papillary mucinous neoplasms, 43 mucinous cystic neoplasms, and 36 non-mucinous cysts) from patients undergoing surgery at 4 tertiary referral centers (January 2010 through June 2017). Histology findings from surgical samples, analyzed independently and centrally re-reviewed in a blinded manner, were used as the reference standard. High-risk PCLs were those with invasive carcinomas, high-grade dysplasia, or intestinal-type intraductal papillary mucinous neoplasms with intermediate-grade dysplasia. An ELISA with Das-1 was performed in parallel using banked cyst fluid samples. We evaluated the biomarker's performance, generated area under the curve values, and conducted multivariate logistic regression using clinical and pathology features. RESULTS: The ELISA for Das-1 identified high-risk PCLs with 88% sensitivity, 99% specificity, and 95% accuracy, at a cutoff optical density value of 0.104. In 10-fold cross-validation analysis with 100 replications, Das-1 identified high-risk PCLs with 88% sensitivity and 98% specificity. The Sendai, Fukuoka, and American Gastroenterological Association guideline criteria identified high-risk PCLs with 46%, 52%, and 74% accuracy (P for comparison to Das-1 ELISA <.001). When we controlled for Das-1 in multivariate regression, main pancreatic duct dilation >5 mm (odds ratio, 14.98; 95% confidence interval, 2.63-108; P < .0012), main pancreatic duct dilation ≥1 cm (odds ratio, 47.9; 95% confidence interval, 6.39-490; P < .0001), and jaundice (odds ratio, 6.16; 95% confidence interval, 1.08-36.7; P = .0397) were significantly associated with high-risk PCLs. CONCLUSIONS: We validated the ability of an ELISA with the monoclonal antibody Das-1 to detect PCLs at risk for malignancy with high levels of sensitivity and specificity. This biomarker might be used in conjunction with clinical guidelines to identify patients at risk for malignancy.

Transmembrane mucin MUC13 distinguishes intraductal papillary mucinous neoplasms from non-mucinous cysts and is associated with high-risk lesions.

BACKGROUND: Intraductal papillary mucinous neoplasms (IPMN) are currently managed based on imaging characteristics and cyst fluid sampling. This study was designed to determine if MUC13, a glycoprotein aberrantly overexpressed in pancreatic adenocarcinoma, might aid in distinguishing high-risk lesions (high grade dysplasia/invasive disease) from low-grade lesions. METHODS: MUC13 immunohistochemical staining was performed on surgically resected formalin-fixed tissue specimens from 49 IPMNs and 23 non-mucinous cysts. Membranous MUC13 expression was measured by H-score, which quantifies staining intensity and the percentage of cells involved (range 0-300). RESULTS: MUC13 expression was detected in all IPMNs and was significantly greater than in non-mucinous cysts (median 210 vs 40, p < 0.001). MUC13 expression was similar among main (n = 26), branch (n = 15), and mixed (n = 8) duct lesions (median 210, 200, 225, respectively). The highest expression was observed in tumors with intestinal and pancreatobiliary histologic features (both median 225) and the lowest in gastric type lesions (median 200). MUC13 expression was significantly greater in high-risk lesions (n = 21) compared to those with low-grade dysplasia (n = 28) (median 250 vs 195, p < 0.001). CONCLUSION: MUC13 expression was significantly greater in high-risk IPMNs in this analysis. The preoperative assessment of MUC13 in cyst fluid samples warrants further investigation.

Highly Accurate Identification of Cystic Precursor Lesions of Pancreatic Cancer Through Targeted Mass Spectrometry: A Phase IIc Diagnostic Study.

Purpose Pancreatic cystic lesions are common incidental findings on imaging, but up to half may be forerunners of pancreatic cancer. Therefore, accurate differential diagnosis is crucial for correct patient management. Unfortunately, currently available diagnostic methods cannot robustly identify premalignant and malignant pancreatic cystic lesions. Methods Cyst fluid samples obtained by routine endoscopic ultrasound-guided aspiration were used for the analyses. In a cohort of 24 patients, eight biomarker candidates for malignant potential and high-grade dysplasia/cancer were identified by an explorative proteomic approach. Subsequently, a quantitative analysis, using 30 heavy-labeled peptides from the biomarkers and parallel reaction monitoring mass spectrometry, was devised, tested in a training cohort of 80, and prospectively evaluated in a validation cohort of 68 patients. End points were surgical pathology diagnosis/clinical follow-up. Diagnostic assessments were blinded to mass spectrometry results. Results The optimal set of markers for detecting malignant potential was a panel of peptides from mucin-5AC and mucin-2, which could discriminate premalignant/malignant lesions from benign with an accuracy of 97% (95% CI, 89% to 99%) in the validation cohort. This result compared favorably with the accuracy of standard analyses: cyst fluid carcinoembryonic antigen (61%; 95% CI, 46% to 74%; P < .001) and cytology (84%; 95% CI, 71% to 92%; P = .02). A combination of proteins mucin-5AC and prostate stem-cell antigen could identify high-grade dysplasia/cancer with an accuracy of 96% (95% CI, 90% to 99%), and detected 95% of malignant/severely dysplastic lesions, compared with 35% and 50% for carcinoembryonic antigen and cytology ( P < .001 and P = .003, respectively). Conclusion Targeted mass spectrometry analysis of just three cyst fluid biomarkers provides highly accurate identification and assessment of cystic precursors to pancreatic adenocarcinoma. Additional studies should determine whether the method can facilitate timely cancer diagnosis, successful intervention, and prevention.

Proteome characterization of human pancreatic cyst fluid from intraductal papillary mucinous neoplasm by liquid chromatography/tandem mass spectrometry.

RATIONALE: In recent years, the molecular components of pancreatic cyst fluid have been used for diagnosis and prognosis. Because the protein markers that are currently used in clinical tests are unreliable, proteomic studies to find new protein markers are being conducted. However, such researches have been limited due to the complexity of pancreatic cyst fluid and the immaturity of proteomic techniques. METHODS: To overcome these limitations and provide a pancreatic cyst proteome dataset, we examined cyst fluid proteome with tandem mass spectrometry. The proteomic analysis was performed using a Orbitrap-based mass spectrometer (Q-Exactive) coupled with a 50-cm-long nano-liquid chromatography column. Protein mutations were identified using mutation sequence database search. RESULTS: A total of 5850 protein groups were identified from microliters of cyst fluid. Among those, 3934 protein groups were reported for the first time in pancreatic cyst fluid. Although high-abundance proteins were not depleted in the experiment, our dataset detected almost all pancreatic tumor markers such as mucin family members, S100 proteins, and CEA-related proteins. In addition, 590 protein mutation marker candidates were discovered. CONCLUSIONS: We provide a comprehensive cyst proteome dataset that includes cystic cellular proteins and mutated proteins. Our findings would serve as a rich resource for further IPMN studies and clinical applications. The MS data have been deposited in the ProteomeXchange with identifier PXD005671 (http://proteomecentral.proteomexchange.org/dataset/PXD005671).

Differentiation of Pancreatic Cyst Types by Analysis of Rheological Behavior of Pancreatic Cyst Fluid.

Differentiation between mucinous and non-mucinous pancreatic cysts is exceedingly important and challenging, particularly as the former bears malignant transformation potential. Pancreatic cyst fluid (PCF)-based diagnostics, including analyses of biochemical markers, as well as cytology, has shown inadequate accuracy. Herein, a preliminary single-center study of 22 PCF samples, collected by endoscopic ultrasound-guided fine needle aspiration (EUS-FNA), assessed the rheological behavior of PCF and its correlation with lesion type. The dependence of PCF shear viscosity on shear rate was found to follow a power law and could be fitted using Ostwald-de Waele model. Three types of flow curves were identified, where two types correlated with non-mucinous cysts, differing by their power law exponent, and the third type corresponding to mucinous cysts. Viscosity measured at a high shear rate was shown to serve as an accurate and independent marker distinguishing between mucinous and non-mucinous cysts, with an optimal cutoff value of ηc = 1.3 cP The accuracy of this novel technique proved superior to string-sign, cytology, carcinoembryonic antigen, and amylase assessments. Moreover, the combined predictive value of ηc and patient age provided for sensitivity and specificity of 100% and 95.5%, respectively. This simple and rapid diagnostic tool can be immediately implemented after EUS-FNA sampling.

Role of biochemistry and cytological analysis of cyst fluid for the differential diagnosis of pancreatic cysts: A retrospective cohort study.

BACKGROUND: Management of pancreatic cysts is based on neoplastic-nonneoplastic discrimination. Endoscopic ultrasound (EUS) enables to differentiate neoplastic-nonneoplastic lesions and also allows fine-needle aspiration (FNA). In this study, we aim to assess feasibility and clinical relevance of cytological and biochemical analysis in differential diagnosis of cystic pancreatic lesions in patients who had undergone endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) due to pancreatic cysts. METHODS: Participants were 96 patients who had undergone EUS-FNA for differential diagnosis of pancreatic cysts. Pancreatic cysts were classified as benign-mucinous, nonmucinous, and malignant according to patient history, physical examination, EUS appearance, and cystic fluid assessment. Tumor markers (CEA, CA(cancer antigens) 72.4, CA 19-9) , amylase, lipase and cytological assesment were compared between 3 different groups. Receiver-operating characteristics (ROC) curves were constructed to identify appropriate cut-off values. RESULTS: Fluid CEA and CA 72.4 levels for benign-mucinous and malignant cysts were significantly higher than for nonmucinous cysts (P ≤ 0.04). A cut-off CEA level of 207 ng/mL differentiated mucinous etiology with a sensitivity of 72.7%, specificity of 97.7%, and accuracy of 89.5%. The sensitivity, specificity, and accuracy of the CA 72.4 cut-off level of 3.32 ng/mL were 80%, 69.5%, and 73.6%, respectively. CONCLUSION: Cyst fluid CEA and CA 72.4 levels have a high accuracy in discriminating mucinous from nonmucinous cysts. When combined with cytology their accuracy rate increases.

Pancreatic Lymphoepithelial Cyst Showing Multiple Floating Ball-like Appearances.

A lymphoepithelial cyst (LEC) is a rare pancreatic lesion, histologically showing squamous epithelia, dense lymphoid tissues, and a keratin substance. Cross-section images of the pancreatic LEC typically show a well demarcated unilocular or multilocular cyst without a solid component. Here we report a rare case of pancreatic LEC in which multiple floating ball-like components were depicted via endoscopic ultrasound. The ball-like components were also depicted by various imaging methods such as computed tomography (CT) showing low-density components, T1-weighted magnetic resonance imaging (MRI) and diffusion-weighted imaging (DWI) showing high-intensity components, and T2-weighted MRI showing low-intensity components. The ball-like components in all images were not well enhanced. Laparotomic cyst resection was performed, and the surgical material revealed keratin balls inside the pancreatic LEC. Keratin components of a pancreatic LEC can take a liquid, sludge, or solid form. Clinicians must be aware of the variations in imaging to facilitate the differentiation and management of pancreatic cystic lesions.

Quantitative real-time detection of carcinoembryonic antigen (CEA) from pancreatic cyst fluid using 3-D surface molecular imprinting.

In this study, a sensitive, yet robust, biosensing system with real-time electrochemical readout was developed. The biosensor system was applied to the detection of carcinoembryonic antigen (CEA), which is a common marker for many cancers such as pancreatic, breast, and colon cancer. Real time detection of CEA during a medical procedure can be used to make critical decisions regarding further surgical intervention. CEA was templated on gold surface (RMS roughness ∼3-4 nm) coated with a hydrophilic self-assembled monolayer (SAM) on the working electrode of an open circuit potentiometric network. The subsequent removal of template CEA makes the biosensor capable of CEA detection based on its specific structure and conformation. The molecular imprinting (MI) biosensor was further calibrated using the potentiometric responses in solutions with known CEA concentrations and a detection limit of 0.5 ng ml(-1) was achieved. Potentiometric sensing was then applied to pancreatic cyst fluid samples obtained from 18 patients when the cyst fluid was also evaluated using ELISA in a certified pathology laboratory. Excellent agreement was obtained between the quantitation of CEA obtained by both the ELISA and MI biosensor detection for CEA. A 3-D MI model, using the natural rms roughness of PVD gold layers, is presented to explain the high degree of sensitivity and linearity observed in those experiments.

Cystic fluid chromogranin A levels in different pancreatic cystic lesions.

BACKGROUND/AIMS: Pancreatic cystic lesions have a broad spectrum of differential diagnosis. There is an ongoing demand to identify specific and sensitive cystic fluid markers for the differential diagnosis of pancreatic cysts. We aimed to evaluate the diagnostic value of cystic fluid chromogranin A (CgA) in the differential diagnosis of pancreatic cysts. MATERIALS AND METHODS: Patients who underwent endoscopic ultrasound (EUS)-guided aspiration for pancreatic cysts were included in the study. Cytopathological analysis and biochemical analysis, including cystic fluid carcinoembryonic antigen (CEA), amylase, Ca 19-9, and CgA, were performed. RESULTS: Fifty-three patients were included in the study. The final diagnosis of patients was 14 pancreatic pseudocysts, 10 intraductal papillary mucinous neoplasms (IPMNs), 8 mucinous cystic neoplasms (MCN), 8 serous cystadenomas (SCAs), 2 cystic pancreatic neuroendocrine tumors (PNETs), and 11 others. The mean CgA levels were 50.51±130.04 ng/mL in pseudocysts, 12.38±8.59 in MCN, and 13.76±10.90 in cystic PNET. There was only one patient with a very high cystic fluid CgA (515.49 ng/mL) and was diagnosed as pseudocyst developed in chronic pancreatitis patient. Two patients with cystic PNET had normal levels of cystic fluid CgA. CONCLUSION: Cystic fluid CgA is not a useful marker for the differential diagnosis of cystic PNETs. It also has no value in the differential diagnosis of other pancreatic cysts.