Tu1680 MAnagement of Patients With Pancreatic Cysts Using Integrated Molecular Pathology

Su1498 In Vivo Characterization of Mucinous Cystadenomas by Needle-Based Confocal Laser Endomicroscopy (nCLE)

Diagnosis of Pancreatic Cystic Lesions by Virtual Slicing: Comparison of Diagnostic Potential of Needle-Based Confocal Laser Endomicroscopy versus Endoscopic Ultrasound-Guided Fine-Needle Aspiration

Benign Pancreatic Cystic Neoplasms: Does F/U EUS-FNA Confirm or Alter the Diagnosis

Pancreatic cystic lesions include inflammatory pseudocysts, benign serous cystadenomas, and mucinous neoplasms, some of which are malignant. Cytologic analysis of cyst fluid has been proposed to diagnose pancreatic cysts before definitive therapy. The authors report an analysis of 31 pancreatic cyst aspirates: 9 pseudocysts, 5 serous cystadenomas, 8 mucinous cystic neoplasms, 4 mucinous cystadenocarcinomas, 2 papillary cystadenocarcinomas, 1 mucinous ductal adenocarcinoma with cystic degeneration, and 2 cystic islet cell tumors. All pseudocysts were correctly classified as probable inflammatory lesions, because of the presence of abundant acute inflammation and histiocytes and the absence of glandular epithelium. Three of five serous cystadenomas were correctly classified, based on the presence of small cuboidal cells in clusters with microvesicular cytoplasm containing glycogen. Eleven of 12 mucinous tumors contained round cells with large cytoplasmic mucin vacuoles or columnar cells containing cytoplasmic mucin. Malignancy was diagnosed in 5 of 7 carcinomas, 1 case was classified as suspicious for malignancy, and 1 case was nondiagnostic because of the absence of a cellular component. The authors concluded that pancreatic cyst fluid cytologic analysis is useful in differentiating mucinous from nonmucinous pancreatic cysts and may provide definitive evidence of malignancy. In some cases, serous cystadenoma can be diagnosed based on cytologic analysis. An inflammatory smear without epithelial cells suggests a pseudocyst, but these findings are nonspecific, as a similar pattern may occur when a cystic neoplasm undergoes degenerative changes. Therefore, pseudocyst remains a diagnosis of exclusion.

*Objective: Patients with potentially premalignant neoplastic pancreatic cysts without high-risk stigmata usually enter a surveillance program. Data on outcomes of such surveillance programs are scarce. We aimed to evaluate the resection rate and malignancy rate during follow-up.Material and methods: From our prospective database (2006–2015) of patients with pancreatic cysts, we analyzed patients with pancreatic cysts without high-risk stigmata with at least six months follow-up.Results: In total, 146 patients were followed for a median of 29 months (IQR 13.5–50 months). In 124 patients (84.9%), no changes in clinical or imaging characteristics occurred during follow-up. Thirteen patients (8.9%) developed an indication for surgery after a median follow-up of 25 months (IQR 12–42 months). Two patients did not undergo surgery because of comorbidity, 11 patients (7.5%) underwent resection. Indications for surgery were symptoms (n = 2), development of a pancreatic mass (n = 1), a new nodule (n = 2), thickened cyst wall (n = 1), pancreatic duct dilation (n = 3), and/or suspicion of mucinous cystic neoplasm (MCN) (n = 3). Postoperative histology showed one pancreatic malignancy not originating from the cyst, three mixed type-intraductal papillary mucinous neoplasm (IPMN), one side branch-IPMN, two MCN, one neuroendocrine tumor, one serous cystadenoma, one inflammatory cyst, and one lymphangioma. The highest grade of cyst dysplasia was borderline dysplasia.Conclusions: Most neoplastic pancreatic cysts without high-risk stigmata at initial presentation show no substantial change during 1–4-year follow-up. Only 7.5% of patients underwent surgery and less than 1% of patients developed pancreatic malignancy. This indicates that additional markers are needed to tailor treatment of pancreatic cysts.

Purpose: Discriminating benign from premalignant cysts of the pancreas is limited by the performance of cyst fluid aspirate and cytology. The presence of KRAS mutations in cyst fluid has been reported to increase the yield of cyst fluid aspirate for the presence of neoplastic cysts. Recently, mutations in GNAS have been found to have an oncogenic role in intraductal papillary mucinous neoplasms (IPMN) through promotion of adenyl cyclase activity and cAMP levels. Our aim was to evaluate whether analysis of cyst fluid DNA obtained by EUS for GNAS mutations can enhance the preoperative diagnosis of neoplastic pancreatic cysts. Methods: Endoscopic ultrasound-guided pancreatic cyst aspirates were collected during a 24-month period and investigated for cytology, carcinoembryonic antigen (CEA) level, and molecular analysis. KRAS and GNAS point mutations were detected by dideoxy chain termination sequencing (Applied Biosystems) of extracted DNA (qiagen) from cyst fluid. The cyst fluid analysis was compared to the final surgical pathology or cytology. Results: We identified 23 patients (11 male, mean age = 68.2 ± 13.8 [SD] years) with pancreatic cysts who underwent EUS-guided cyst aspiration. The mean cyst diameter was 2.3 cm (range 1-8 cm). Cysts were located in the head (n=10), body (n=8), and tail (n=5) of the pancreas. The cohort consisted of four serous cystadenomas (SCA), three mucinous cyst neoplasms (MCN), one pseudocyst, five solid-cystic adenocarcinoma (PDA), two solid-cystic endocrine neoplasms, one lymphoepithelial cyst, and seven IPMNs. The mean cyst fluid CEA levels in IPMNs was 3,895 ng/mL (range 22-24,747); 3/7 patients with IPMN had CEA levels <192 ng/mL. Mutations in KRAS were present in 5/7 (71%) IPMNs; LOH was demonstrated in three of these. Three of seven (43%) IPMNs carried a mutation in GNAS, all of which harbored degrees of dysplasia. The mutations in GNAS occurred at codon 201. All IPMNs carried a mutation in at least one of the two oncogenes. No mutations were identified in the SCA, in contrast to the IPMNs (p<0.01). GNAS mutations were not identified in any of the MCNs or PDA (p<0.005 when compared to IPMNs), although KRAS mutations were found in 1/3 (33%) of MCNs and 3/5 (60%) of PDAs. Cyst fluid CEA >192 ng/mL had a sensitivity of 57% to predict the presence of an IPMN. The sensitivity of KRAS mutation in cyst fluid was 71% for the diagnosis of IPMN, but with the addition of GNAS mutations, all IPMNs were identified (sensitivity = 100%, p<0.05). Conclusion: The presence of GNAS in combination with KRAS in pancreatic cystic fluid improves the sensitivity for diagnosis of IPMN when compared to CEA or KRAS alone. This demonstrates the feasibility of GNAS analysis from pancreatic cyst fluid obtained by EUS guided aspiration.

This study aimed to determine the distribution of etiology of pancreatic cysts using established criteria/markers from cyst fluid analysis and cytology that have been reported to have high specificity in published literature. A retrospective study of pancreatic cysts using an endoscopic database from March 2002 and May 2013 was conducted. Pancreatic cysts <10 mm and cysts with a history of pancreatic cancer were excluded. In our cohort of 758 patients with pancreatic cyst(s), the cyst etiology was as follows: mucinous cyst/side-branch intraductal papillary mucinous neoplasms (SB-IPMNs)/mucinous cystic neoplasms (MCN; 48.2%), pseudocyst (27.6%), serous cystadenoma (11%), simple cysts (6.4%), mucinous cystadenocarcinoma (5.1%), and other (1%). Approximately 41% (n = 310) of the cysts were ≥3 cm in size and included the following: pseudocyst (39.7%), mucinous cysts/SB-IPMN/MCN (28.1%), serous cystadenoma (16.7%), mucinous cyst adenocarcinoma (9.7%), and simple cyst (4.8%). In 118 patients with a known history of acute pancreatitis, the cyst diagnoses included pseudocyst (68.7%), mucinous cyst/SB-IPMN/MCN (18.6%), benign/simple cyst (7.6%), and mucinous cystadenocarcinoma (2.5%). In patients with cystic pancreatic lesion noted on cross-sectional imaging, approximately half of the patients have lesions without malignancy or malignant potential and therefore not requiring surveillance. Endoscopic ultrasound/endoscopic ultrasound-guided fine-needle aspiration evaluation of the pancreatic cysts can help optimize their further management.

Diagnosis and Management of Pancreatic Cysts

A 32-year-old Western-Indian woman presented with a history of chronic abdominal pain dating back to her previous pregnancy and had previously been told that she had a pancreatic pseudocyst. She was admitted later to our hospital with abdominal pain, nausea, and vomiting in addition to anorexia for the past week. Her medical history was significant for 2 cesarean sections followed by tubal ligations. She was taking multiple pain medications. She denied tobacco use but admitted occasional alcohol consumption. Her family history was positive for colon cancer and diabetes mellitus. On physical examination, she appeared well nourished, although in mild distress, and her vital signs were stable. The abdomen was soft and slightly distended, with no tenderness. A laboratory workup was performed, which included a complete blood count, electrolytes, and liver and kidney function tests in addition to amylase and lipase; all were within reference ranges. A previous abdominal ultrasound examination followed by an endoscopic retrograde cholangiopancreatography showed a 5-cm pancreatic cyst. A computed tomographic scan of the abdomen with contrast showed a 3.0-cm nonenhancing cyst in the body of the pancreas (Figure 1). The clinical impression was that of chronic pancreatitis with a pancreatic pseudocyst. Distal pancreatectomy was performed. Gross examination of the tissue specimen revealed a 2.1 × 1.9 × 1.6-cm unilocular cyst with a smooth inner lining containing tan mucoid material. The remainder of the pancreas appeared unremarkable. Extensive sampling of the cyst showed essentially similar microscopic features from one field to another (Figures 2 and 3).What is your diagnosis?Mucinous cystic neoplasms of the pancreas are usually multicystic and lined by mucinous epithelium, which is supported by ovarian-like cellular stroma, and they occur predominantly in middle-aged women. The mean age at diagnosis is 49 years, and the female-male ratio is 9:1.1–5 By definition, they are not connected to the pancreatic duct system.1 The presence of ovarian-like stroma and the absence of connection to the pancreatic duct system separate them from intraductal papillary mucinous neoplasms. They were first recognized as a distinct clinicopathologic entity in 1978 by Compagno and Oertel.1 Clinically, mucinous cystic neoplasms are usually asymptomatic and discovered incidentally during radiologic workup for other reasons.67 When symptoms are present, they are usually of the nonspecific upper abdominal type unless the cyst is large with compression to adjacent structures. The predominant location is the tail or the body of the pancreas.6 Radiologically, mucinous cystic neoplasms exhibit large cysts averaging between 6 and 10 cm with thick capsules and occasionally solid areas.145 Grossly, the tumor is characteristically well circumscribed and unicystic (occasionally multicystic) and contains thick viscous mucin, which is surrounded by a thick capsule that may contain calcifications. Microscopic examination usually reveals a single layer of mucinous epithelium, which is occasionally flattened or even denuded. Therefore, extensive sampling is recommended to rule out the possibility of a pancreatic pseudocyst or a more complex, often higher-grade mucinous cystic neoplasm.Typically, an ovarian-like stroma supports the epithelium, which may express estrogen and progesterone receptors. The mechanism and pathophysiology of this unusual phenomenon remain uncertain. The epithelium may form pseudopapillae; however, further cytologic or architectural atypia is not common in the adenoma type.8Distinction of the adenoma from higher-grade mucinous cystic neoplasms requires the presence of true papillae with pseudostratification and true stratification, mild-to-moderate cytologic atypia, and mitoses. If these features are present, they are best termed mucinous cystic neoplasms with low malignant potential or mucinous cystic neoplasms, borderline type. Those exhibiting severe cytologic atypia/carcinoma in situ, loss of mucin-secreting features, more abundant mitoses, tufting, and marked stratification with no evidence of stromal invasion are described as noninvasive mucinous cystic carcinomas. Finally, the presence of true stromal invasion further distinguishes the invasive variant.9 Mucinous cystic neoplasms are usually treated by excision, and the prognosis depends on the completeness of excision as well as the presence or absence of carcinoma.6 Mucinous cystic neoplasms with invasive carcinoma harbor a significantly better prognosis than pancreatic ductal adenocarcinomas.10 The differential diagnosis of mucinous cystic neoplasms includes a pancreatic pseudocyst and all other cystic tumors; however, a thorough gross examination and an extensive sampling with radiologic and clinical correlation in conjunction with the aforementioned microscopic features allow the distinction. A recent review of pancreatic cystic neoplasms by Goldsmith8 is very helpful and is highly recommended.

The authors evaluated cyst fluid CA 72-4 as a tumor marker in the differential diagnosis of pancreatic cystic lesions. Pancreatic cystic lesions include inflammatory pseudocysts, serious cystadenomas, and mucinous tumors. Mucinous tumors can be further subdivided into mucinous cystadenocarcinomas and premalignant mucinous cystic neoplasms. The clinical and radiologic features of these lesions are unreliable to make a preoperative diagnosis of these diagnostically difficult lesions. Analysis of aspirated cyst fluid was proposed as an aid to making the preoperative differential diagnosis. Currently, a number of parameters have been reported as useful markers in cyst fluid aspirates, including the tumor markers carcinoembryonic antigen and CA 15.3, enzymes (amylase, lipase, and amylase isoenzymes), relative viscosity, and cytologic analysis. However, owing to the rarity of pancreatic cystic tumors, experience with cyst fluid analysis is limited. To define additional markers that might be useful in the differential diagnosis of pancreatic cysts, the authors measured the tumor-associated glycoprotein 72 (TAG-72) in aspirates from 19 pancreatic cystic lesions. Cyst fluid from 19 pancreatic cysts was obtained by needle aspiration. The tumor marker TAG-72 was measured by a commercial (CA 72-4) immunoassay. Cyst fluid CA 72-4 levels in mucinous cystadenocarcinomas were markedly elevated (mean, 10,027 U/mL; range, 780 to 34,853 U/mL) compared with that in pseudocysts (mean, 3.8 U/mL; range, < 3 to 5.7 U/mL) and serous cystadenomas (mean and range, < 3 U/mL; p < 0.001). The level of CA 72-4 in benign mucinous cystic neoplasms was intermediate (mean, 44.2 U/mL; range, < 3 to 137 U/mL), but it was statistically different from either carcinomas (p = 0.009) or benign cysts (p < 0.001).

Background: Current preoperative differentiation of premalignant and malignant pancreatic cysts from benign pancreatic cysts based on imaging and cyst fluid analysis is inadequate. This inability to accurately differentiate has resulted in unnecessary surgical resections. This is the first study applying proteomic technology to identify novel proteins or protein patterns in pancreatic cyst fluid which may more accurately predict pancreatic cyst pathology preoperatively and help triage patient care. Methods: Pancreatic cyst fluid was collected by endoscopic ultrasound guided FNA from patients with pathologically confirmed pancreatic mucinous and non-mucinous cysts, centrifuged and stored immediately at −80°C. Fluid from representative pancreatic cysts was prepared for proteomic analysis using centrifugation, acetone, ethanol and TCA precipitation, followed by solubilization into highly chaotropic nonionic detergents and buffer for 2-D electrophoresis and 2 dimensional liquid chromatography (PF2D: 1-D chromatofocussing, 2D-reverse phase). Spots from 2D gels were robotically excised, digested and mass spectrometry was used to identify unique differential proteins using liquid chromatography tandem mass spectrometry (LC-MS/MS) and MALTI-TOF. Proteins were identified using Mascot search engine and Sequest search algorithms. Results: 122 pancreatic cyst fluid samples were collected, stored and processed according to the protocol. Initial protein quantification for ranged from 1 to 5 mg/ml. Pancreatic cyst fluid from 5 samples (2 mucinous and 3 serous cystadenomas) was used for initial proteomic analysis (2D gel electrophoresis (2DE:(3)), PF2D(2)). Initial analysis shows a striking differential protein profile between serous and mucinous samples (by PF2D) in both dimensions (1-D chromatofocussing and 2-D reverse phase). Comparison of two-dimensional maps of the pancreatic serous and mucinous cystadenomas show few overlaps thus suggesting many proteins unique to each cystic neoplasm. Interpretable 2-D electrophoresis gels were obtained from pancreatic cyst fluid. Using 2-D Electrophoresis we have already identified over 20 unique cellular and membrane proteins using LC-MS/MS. Through 2-D electrophoresis gel we have identified cellular and membrane proteins using LC-MS/MS. Chromatofocussed fractions were processed over a second dimension HPRP column to produce over 700 fractions in each experiment. The second dimension reverse phase fractions for serous cyst show a very different protein profile than mucinous cysts. Conclusions: To our knowledge, this is the first report detailing the proteome of pancreatic cystic neoplasm fluid. Differential protein expression was found between pancreatic mucinous and serous cystadenoma fluid. Identification of specific protein patterns or proteins may be used as highly accurate serum based or cyst fluid based biomarkers.

This study was performed to evaluate the utility of serum and cyst fluid analysis for enzymes (amylase and lipase) and tumor markers (carcinoembryonic antigen, CA 19-9, CA 125, and CA 72-4) in the differential diagnosis of cystic pancreatic lesions. Serum and cyst fluid were obtained from 48 patients with pancreatic cysts (21 pseudocysts, 14 mucinous cystic neoplasms, 6 ductal carcinomas, and 7 serous cystadenomas), observed between 1989 and 1994. Serum CA 19-9 levels were significantly higher in ductal carcinomas (all > 100 U/mL) and mucinous cystic neoplasms (P < 0.05). CA 72-4 cyst fluid levels were significantly higher in mucinous cystic tumors (P < 0.005), with 95% specificity and 80% sensitivity in detecting mucinous or malignant cysts. A combined assay of serum CA 19-9 and cyst fluid CA 72-4 correctly identified 19 of 20 (pre-) malignant lesions (95%), with only 1 false-positive result (3.6%). Cytology showed a sensitivity of 48% and specificity of 100%. Any pancreatic cyst with high serum CA 19-9 values, positive cytology, or high CA 72-4 in the fluid should be considered for resection.

One concern associated with pancreatic diseases is the poor prognosis of pancreatic cancer. Even with advances in diagnostic modalities, risk stratification of premalignant lesions and differentiation of pancreatic cysts are challenging. Pancreatic lesions of concern include intraductal papillary mucinous neoplasms, mucinous cystic neoplasms, serous cystadenomas, pseudocysts, and retention cysts, as well as cystic degeneration of solid tumors such as solid pseudopapillary neoplasms and pancreatic neuroendocrine neoplasms. Pancreatic juice obtained during endoscopic retrograde cholangiopancreatography has previously been used for the detection of KRAS mutation. Recently, duodenal fluid, which can be obtained during the relatively minimally invasive procedures of endoscopic ultrasound (EUS) and esophagogastroduodenoscopy, and cyst fluid collected by EUS-guided fine-needle aspiration (FNA) were used for molecular biological analysis. Furthermore, advanced analytic methods with high sensitivity were used for the detection of single and multiple markers. Early detection of malignant pancreatic tumors and risk stratification of premalignant tumors can be performed using duodenal fluid samples with a single marker with high sensitivity. Technological advances in simultaneous detection of multiple markers allow for the differentiation of cystic pancreatic tumors. One thing to note is that the clinical guidelines do not recommend pancreatic cyst fluid and pancreatic juice (PJ) sampling by EUS-FNA and endoscopic retrograde cholangiopancreatography, respectively, in actual clinical practice, but state that they be performed at experienced facilities, and duodenal fluid sampling is not mentioned in the guidelines. With improved specimen handling and the combination of markers, molecular markers in PJ samples may be used in clinical practice in the near future.

American Gastroenterological Association guidelines are inaccurate in detecting pancreatic cysts with advanced neoplasia: a clinicopathologic study of 225 patients with supporting molecular data

Mucinous pancreatic cysts (intraductal papillary mucinous neoplasm and mucinous cystic neoplasm) have the potential to progress to invasive pancreatic adenocarcinoma, presenting an opportunity for early detection, prevention, and cure. Serous cystic neoplasms (SCN) have no malignant potential, but can mimic mucinous pancreatic cysts on imaging. Therefore, identification of biomarkers that can distinguish between cystic lesions is critically important. We hypothesize that vascular endothelial growth factor (VEGF)-A levels in pancreatic fluid correlate with pathologic diagnosis. Pancreatic cyst/duct fluid samples were prospectively collected from patients undergoing pancreatic resection and correlated with surgical pathology. VEGF levels were detected by ELISA. VEGF-A and VEGF receptor 2 expression in pancreatic tissue was localized by immunohistochemistry. Genetic alterations of the von Hippel-Lindau gene were determined by targeted next-generation sequencing. Eighty-seven patients met inclusion criteria for enrollment. Final pathologic diagnoses included pseudocyst (n = 9), SCN (n = 17), mucinous cystic neoplasm (n = 24), low/moderate grade intraductal papillary mucinous neoplasm (n = 16), high-grade/invasive intraductal papillary mucinous neoplasm (n = 10), and pancreatic ductal adenocarcinoma (n = 11). VEGF-A was significantly upregulated in SCN cyst fluid compared with all other diagnoses (p < 0.0001). With a cut-off of 8,500 pg/mL, VEGF-A has 100% sensitivity and 97% specificity as an SCN biomarker. VEGF-A and VEGF receptor 2 are overexpressed in SCN cyst tissue. VEGF-C was also significantly elevated in SCN cyst fluid (p < 0.0001). With a cut-off set at 200 pg/mL, VEGF-C identifies SCN with 100% sensitivity and 90% specificity. The presence of a von Hippel-Lindau mutation in SCN cyst tissue correlates with elevated cyst fluid VEGF levels. This is the first report of a cyst fluid protein biomarker that can positively identify SCN. The ability to distinguish SCN from premalignant/malignant pancreatic cysts can spare the cost and risk of surveillance and surgical intervention in select patients.