Global Protease Activity Profiling Provides Differential Diagnosis of Pancreatic Cysts.

Purpose: Pancreatic cysts are estimated to be present in 2%-3% of the adult population. Unfortunately, current diagnostics do not accurately distinguish benign cysts from those that can progress into invasive cancer. Misregulated pericellular proteolysis is a hallmark of malignancy, and therefore, we used a global approach to discover protease activities that differentiate benign nonmucinous cysts from premalignant mucinous cysts.Experimental Design: We employed an unbiased and global protease profiling approach to discover protease activities in 23 cyst fluid samples. The distinguishing activities of select proteases was confirmed in 110 samples using specific fluorogenic substrates and required less than 5 µL of cyst fluid.Results: We determined that the activities of the aspartyl proteases gastricsin and cathepsin E are highly increased in fluid from mucinous cysts. IHC analysis revealed that gastricsin expression was associated with regions of low-grade dysplasia, whereas cathepsin E expression was independent of dysplasia grade. Gastricsin activity differentiated mucinous from nonmucinous cysts with a specificity of 100% and a sensitivity of 93%, whereas cathepsin E activity was 92% specific and 70% sensitive. Gastricsin significantly outperformed the most widely used molecular biomarker, carcinoembryonic antigen (CEA), which demonstrated 94% specificity and 65% sensitivity. Combined analysis of gastricsin and CEA resulted in a near perfect classifier with 100% specificity and 98% sensitivity.Conclusions: Quantitation of gastricsin and cathepsin E activities accurately distinguished mucinous from nonmucinous pancreatic cysts and has the potential to replace current diagnostics for analysis of these highly prevalent lesions. Clin Cancer Res; 23(16); 4865-74. ©2017 AACR.

Cyst Fluid Telomerase Activity Predicts the Histologic Grade of Cystic Neoplasms of the Pancreas.

PURPOSE: Pancreatic cysts frequently pose clinical dilemmas. On one hand, cysts with high-grade dysplasia offer opportunities for cure, on the other hand, those with low-grade dysplasia are easily over treated. Cyst fluid markers have the potential to improve the evaluation of these cysts. Because telomerase activity is commonly activated in malignant cells, we evaluated the diagnostic performance of cyst fluid telomerase activity measurements for predicting histologic grade. EXPERIMENTAL DESIGN: Telomerase activity was measured using telomerase repeat amplification with digital-droplet PCR in surgically aspirated cyst fluid samples from 219 patients who underwent pancreatic resection for a cystic lesion (184 discovery, 35 validation) and 36 patients who underwent endoscopic ultrasound fine-needle aspiration. Methodologic and clinical factors associated with telomerase activity were examined. RESULTS: Telomerase activity was reduced in samples that had undergone prior thawing. Among 119 samples not previously thawed, surgical cyst fluids from cystic neoplasms with high-grade dysplasia ± associated invasive cancer had higher telomerase activity [median (interquartile range), 1,158 (295.9-13,033)] copies/µL of cyst fluid than those without [19.74 (2.58-233.6) copies/µL; P < 0.001)]. Elevated cyst fluid telomerase activity had a diagnostic accuracy for invasive cancer/high-grade dysplasia of 88.1% (discovery), 88.6% (validation), and 88.2% (merged). Among cysts classified preoperatively as having "worrisome features," cyst fluid telomerase activity had high diagnostic performance (sensitivity 73.7%, specificity 90.6%, accuracy, 86.1%). In multivariate analysis, telomerase activity independently predicted the presence of invasive cancer/high-grade dysplasia. CONCLUSIONS: Cyst fluid telomerase activity can be a useful predictor of the neoplastic grade of pancreatic cysts. Clin Cancer Res; 22(20); 5141-51. ©2016 AACRSee related commentary by Allen et al., p. 4966.

Lkb1 regulates organogenesis and early oncogenesis along AMPK-dependent and -independent pathways.

The tumor suppressor Lkb1/STK11/Par-4 is a key regulator of cellular energy, proliferation, and polarity, yet its mechanisms of action remain poorly defined. We generated mice harboring a mutant Lkb1 knockin allele that allows for rapid inhibition of Lkb1 kinase. Culturing embryonic tissues, we show that acute loss of kinase activity perturbs epithelial morphogenesis without affecting cell polarity. In pancreas, cystic structures developed rapidly after Lkb1 inhibition. In lung, inhibition resulted in cell-autonomous branching defects. Although the lung phenotype was rescued by an activator of the Lkb1 target adenosine monophosphate-activated kinase (AMPK), pancreatic cyst development was independent of AMPK signaling. Remarkably, the pancreatic phenotype evolved to resemble precancerous lesions, demonstrating that loss of Lkb1 was sufficient to drive the initial steps of carcinogenesis ex vivo. A similar phenotype was induced by expression of mutant K-Ras with p16/p19 deletion. Combining culture of embryonic tissues with genetic manipulation and chemical genetics thus provides a powerful approach to unraveling developmental programs and understanding cancer initiation.

Proteases present in some pancreatic cyst fluids may affect mucin immunoassay by degrading antibodies and antigens.

OBJECTIVES: Biomarker detection in pancreatic cyst fluids is of importance to improve the diagnosis of mucinous cystadenoma, a precancerous lesion. However, assay protocols are generally established for serum testing. METHODS: Immunoradiometric assay of gastric M1/MUC5AC mucin was performed on pancreatic cyst fluids with well-characterized monoclonal antibodies. RESULTS: Among 1466 pancreatic cyst fluids tested, about 10% to 15% of samples presented abnormal behaviors: (i) radioactivity measured after immunoradiometric assay much lower than the blank of the assay and (ii) increasing dilution of the fluids leading to apparent increase of M1/MUC5AC concentration. In contrast, none of the 109 hepatic cyst fluids tested presented interference.We demonstrate that some (n = 54) interfering fluids cause mucin degradation as well as antibody degradation. Western blot analysis showed that the C-terminal part of the M1/MUC5AC apomucin is most sensitive to degradation. CONCLUSIONS: The presence of proteases that degrade antibodies as well as mucin may explain the pitfalls observed in 3.6% of the samples. To detect this interference, each fluid has to be systematically tested at 1:100 dilution in the presence of a saturating concentration of M1/MUC5AC mucin standard and in the absence of antiprotease reagents. Detection of interference could prevent false results caused by mucin degradation in situ.

Cyst fluid cytology and chemical features in a case of lymphoepithelial cyst of the pancreas: A rare and difficult preoperative diagnosis.

Most pancreatic cysts (90%) are inflammatory pseudocysts. Approximately 10% of pancreatic cysts are neoplasms, including serous cystadenomas, and mucinous tumors, some of which are malignant. Analysis of pancreatic cyst fluid obtained by percutaneous or endoscopic fine-needle aspiration is increasingly being used for the preoperative diagnosis of pancreatic or peripancreatic cysts. However, cyst fluid chemical and cytologic features in less common types of pancreatic cysts have not been reported. Lymphoepithelial cyst of the pancreas is exceedingly rare, and only occasional individual reports have described cyst fluid findings. We report on a case of lymphoepithelial cyst of the pancreas developing in a middle-aged man. Cyst fluid aspirated under radiological guidance showed elevated levels of carcinoembryonic antigen (CEA), CA19-9, CA 125, and amylase, and a viscosity greater than that of serum. A cell block preparation of a fine-needle aspiration showed tissue fragments with a keratinized squamous lining and a lymphocytic infiltrate in the wall, and abundant background keratinous debris. The cytologic and biochemical findings in this case exhibit similarities to the findings reported in other reports, and may represent a recognizable pattern on cyst fluid analysis.

Cyst fluid analysis in the differential diagnosis of pancreatic cysts. A comparison of pseudocysts, serous cystadenomas, mucinous cystic neoplasms, and mucinous cystadenocarcinoma.

Pancreatic cystic lesions include inflammatory pseudocysts, benign serous tumors, and mucinous neoplasms, some of which are malignant. Clinical and radiologic indices are often inadequate to discriminate reliably among these possibilities. In an attempt to develop new preoperative diagnostic criteria to assist in decisions regarding therapy, the authors have performed cyst fluid analysis for tumor markers (carcinoembryonic antigen: CEA, CA 125, and CA 19.9), amylase content, amylase isoenzymes, relative viscosity, and cytology on 26 pancreatic cysts. The cases included nine pseudocysts, five serous cystadenomas, 4 mucinous cystic neoplasms, 7 mucinous cystadenocarcinomas, and one mucinous ductal adenocarcinoma with cystic degeneration. Carcinoembryonic antigen levels were high (> 367) in all benign and malignant mucinous cysts, but were low (< 23) in the pseudocysts and benign serous cystadenomas, an indication that CEA discriminates between mucinous and nonmucinous cysts (p < 0.0001). Values for CA 125 were high in all malignant cysts, low in pseudocysts, and variable in mucinous cystic neoplasms and serous cystadenomas. Levels of Ca 19.9 were nondiscriminatory. Cyst fluid amylase and lipase content were variable but were generally high in pseudocysts and low in cystic tumors. Amylase isoenzyme analysis was useful to differentiate pseudocysts from cystic tumors. Measurement of the relative viscosity in cyst fluid showed high (> serum viscosity) values in 89% of mucinous tumors and low values (< serum) in all pseudocysts and serous cystadenomas (p < 0.01). Cytologic analysis of cyst fluids was of limited value in differentiating pseudocysts from serous cystadenoma, but in seven of eight mucinous tumors provided useful diagnostic information and correctly classified three of five malignant tumors. The authors conclude that cyst fluid analysis can provide a preoperative classification of these diagnostically difficult lesions. The combination of viscosity, CEA, CA 125, and cytology can reliably distinguish malignant cystic tumors and potentially premalignant mucinous cystic neoplasms from pseudocysts and serous cystadenomas. Amylase content with isoenzyme analysis is useful to identify pseudocysts.

[Non-surgical treatment of cysts and pseudocysts of the pancreas. A study of a series of 33 cases]. / Traitement non chirurgical des kystes et pseudo-kystes du pancréas. Etude d'une série de 33 cas.

Pancreatic cysts and pseudocysts had to be treated by surgery until 15 years ago. Nowadays they can be aspirated (or drained) either endoscopically or by ultrasonic and computed tomographic guided punctures. The aim of this study was to see if these non-surgical treatments were efficient enough among the actual treatments. From 1984 to 1988, 33 patients were admitted in one single institution with a pancreatic cyst or pseudocyst, 22 of which were a consequence on an acute pancreatitis and 11 complicated a chronic pancreatitis. Ten cysts were connected with the Wirsung channel while 11 others were not, but the ERCP failed to give any accurate information on this point in 12 cases. As a first treatment, we abstained of any invasive procedure in 9 cases, 18 were treated by percutaneous aspiration guided by ultrasound and 6 patients underwent surgery. The mean follow-up was 30 months. All the patients who had no treatment remained symptom free; their cysts improved in 5 cases and disappeared in 4 cases. Among the cysts treated by percutaneous aspiration, 4 had to be operated, 5 disappeared, 3 improved and 6 recurred; the percutaneous aspiration obtained a lasting symptoms' relief for these patients, whatever the connection the cyst had with the Wirsung channel. This study suggests that percutaneous aspiration or drainage guided by ultrasound may be a treatment of the highest quality for acute and chronic pancreatitis cystic formations. However, it seems to act upon the symptoms rather than upon the cystic formations themselves.

Management of chronic pancreatitis with pancreas cyst by endoscopic pancreatic prosthesis.

Cases of chronic pancreatitis accompanied by stenosis or dilation of the pancreatic duct were treated by endoscopic placement of an endoprosthesis and drainage. The purpose of the treatment was the alleviation of pain, restoration of extrapancreatic secretion and retardation of the progression of inflammation. The procedure was performed successfully in 9 of 13 patients, using an endoscopically placed pigtail prosthesis in the pancreatic duct. The successfully treated patients consisted of 9 cases of chronic pancreatitis, 2 cases being pancreas divisum. After disappearance of the symptoms and abnormal endoscopic findings, the drainage tubes were removed after a period of 5-12 months. No serious complications were encountered. Following the procedure, an improvement in appetite and increase of 2-13 kg in body weight was recognized. This method yielded satisfactory results in the treatment of chronic pancreatitis.

Characterization of phospholipase A2 activity in aspirates of human pancreatic pseudocysts after isolation by reversed-phase high performance liquid chromatography.

Phospholipase A (PLA) is able to attack membrane phospholipids and thereby plays a putative role in the pathogenesis of pancreatic pseudocysts. We looked for PLA2-like activity in aspirates from human pancreatic pseudocysts. In material originating from one cyst which occurred shortly after an acute pancreatitis attack, hydrolyzing enzymatic activity measured by a sensitive bioassay system for PLA2 activity was found without prior trypsin activation (67 x 10(3) U/min/100 microliters). A biochemical characterization of this hydrolyzing enzymatic activity was provided after resolution of the respective proteins contained in the cyst fluid by HPLC. High hydrolyzing activities were found in correspondence to one specific, early eluting peak. The purified enzyme had pH optima at 3.5 and 6. Addition of EDTA (5 mM) to the test system abolished the enzymatic activity which mirrored the requirement for calcium ions. The activity was optimal at calcium concentrations ranging from 1-2 mM. Higher calcium concentrations reduced the enzymatic activity. The enzyme showed high heat stability. SDS-gel analysis of the peak showed one single band with a molecular weight of about 20,000 Daltons. Our findings demonstrate the possibility of activated, PLA-like activity in human pancreatic pseudocyst fluid. We speculate that an inappropriate activation of this enzyme in peri- or intrapancreatic "fluid collections" could account for pseudocyst formation after an acute pancreatitis attack.

Pancreatic pseudocysts: a biochemical evaluation of proteases and protease inhibitors in plasma.

A biochemical evaluation was performed on plasma from eight patients developing a pancreatic pseudocyst during acute pancreatitis attacks and from six patients with a known pseudocyst. Patients developing an acute pancreatic pseudocyst had high levels of activated trypsin in complex with alpha 1-protease inhibitor, together with a probable activation of the kinin, complement, coagulation and fibrinolytic systems. Profound changes were also seen in several protease inhibitors, indicating consumption of the inhibitors. The changes did, however, not differ from those seen in severe acute pancreatitis attacks in which no pseudocyst developed. Patients with chronic pancreatic pseudocysts had biochemical changes similar to those seen in moderate pancreatitis attacks, without any overt cascade system activation. At convalescence, however, these patients had biochemical signs of leakage from the pancreas and an ongoing proteolytic activity.

Values of carcinoembryonic antigen, elastase 1, and carbohydrate antigen determinant in aspirated pancreatic cystic fluid in the diagnosis of cysts of the pancreas.

The levels of carcinoembryonic antigen (CEA), elastase 1, and carbohydrate antigen determinant (CA 19-9) in the pancreatic cystic fluid and the serum from five patients with cystadenocarcinoma of the pancreas, one patient with retention cyst due to pancreatic carcinoma, three patients with cystadenoma, and eight patients with benign pseudocyst accompanying or following pancreatitis, were determined by immunoassay technique. Fluid from pancreatic cysts was obtained by ultrasonically-guided percutaneous fine-needle aspiration biopsy. The specimens were centrifuged and the supernatant was used for the measurement of CEA, elastase 1, and CA 19-9, while the cell pellet was examined cytologically. The levels of CEA in the aspirated fluid were significantly higher in patients with malignant cysts of the pancreas than in those with benign cystadenomas and pseudocysts. In contrast, the levels of elastase 1 were significantly lower in patients with malignant cysts than in those with benign pancreatic cysts. Although the levels of CA 19-9 were significantly higher in patients with malignant cysts than in those with pseudocysts, the overlap between the values of patients with malignant and benign pancreatic cysts is too great. The serum CA 19-9 was most useful, however, to distinguish an individual patient with malignant cysts of the pancreas from those with benign pancreatic cyst, since there were no significant differences between the levels of serum CEA and elastase 1 in patients with malignant and benign pancreatic cysts. Correct diagnoses were made cytologically in 4 (66.7%) of 6 patients with malignant cysts. In two patients with malignant cyst, in whom no cancer cells were detectable in the aspirated materials, levels of CEA were abnormally high, but high levels of elastase 1 did not occur. Therefore, the combined measurement of CEA and elastase 1 in the aspirated cystic fluid of the pancreas could be used as an aid in diagnosis of malignant cysts of the pancreas.

Agarose electrophoresis and inhibitor tests for isoamylase determination can give complementary clinical information.

We report the presence of an extremely high proportion of "aged" amylase in the serum and cyst fluid of a patient with a pancreatic pseudocyst. A salivary amylase inhibitor test helped us to differentiate these "aged" pancreatic amylases from salivary fractions having a similar electrophoretic mobility.

Isoamylases and their thermolability in serum and cyst fluid from patients with pancreatic pseudocysts.

The isoamylase pattern in serum and the amylase thermolability have been suggested as screening tests for the development of pancreatic pseudocysts. To study whether serum reflects the contents of pseudocysts, we have investigated the isoamylases and their thermolability in cyst fluid and in serum from 13 patients with pancreatic pseudocysts. No significant correlation was found between the contents in serum and cyst fluid with regard to total amylase and isoamylase P2 and P3 or with regard to the thermolability of total amylase and isoamylase P2 and P3. Thus, serum does not reflect the cyst contents of isoamylases or their thermolability. Therefore these serum amylase determinations can hardly be expected to be useful in distinguishing patients developing pseudocysts among patients with pancreatitis.

Aging changes of pancreatic isoamylases and the appearance of "old amylase" in the serum of patients with pancreatic pseudocysts.

Human serum and pancreatic secretions contain at least three isoamylases of pancreatic origin. As determined by gel electrophoresis and saccharogenic amylase assay, P1, P2, and P3 designate isoenzymes with slow, intermediate, and rapid electrophoretic mobilities, respectively. The P1 isoamylase normally accounts fo 80-90% of total amylase activity, P2 for 10-20%, and P3 for 0-4% in both serum and pancreatic juice. When pancreatic amylase is incubated at 37 degrees C, P1 decreases, and P2 and P3 increase within hours. Whereas P2/P1 is always < 0.25 in fresh pancreatic juice, normal serum, acute pancreatitis serum, chronic pancreatitis serum, or pancreatic cancer serum, the ratio was elevated in 11 of 12 pseudocyst contents (mean P2/P1 = 0.51) and in 14 of 16 sera from patients with proven pseudocysts (mean P2/P1 = 0.43) (P < 0.001). After pseudocysts were surgically drained, the proportions of the pancreatic isoamylases in serum reverted to normal. The precise, characteristic, and predictable changes in electrophoretic mobility, presumably a result of specific spontaneous chemical alterations of the isoenzyme molecules, allow identification of "old amylase" in the serum of patients with pancreatic pseudocysts. This finding may be a useful adjunct for diagnosis, but whether the amount of "old amylase" can be used to estimate the age of a pseudocyst is not yet known.

Serial serum amylase levels in patients with pancreatic pseudocysts.

The incidence of hyperamylasaemia in serial serum amylase levels from patients with pancreatic pseudocysts is not well documented in the literature. Accordingly serial amylase levels were estimated in 5 patients with proven pancreatic pseudocysts. The majority of the values were normal or only minimally elevated. In contrast the amylase thermolability was persistently elevated and appeared to be a more sensitive measurement than serum amylase activity for indicating the presence of pancreatic pseudocysts.