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.

Telomerase activity is a useful marker to distinguish malignant pancreatic cystic tumors from benign neoplasms and pseudocysts.

BACKGROUND: Pancreatic serous cystadenoma, mucinous cystic neoplasms, ductal adenocarcinoma with cystic change, and pseudocysts are a spectrum of pancreatic cystic lesions. Their management strategy and prognosis are extremely diverse. Imaging study, cytology, and analysis of the tumor markers of cyst fluid are not always reliable in differentiation of these disease entities. MATERIALS AND METHODS: Fifteen patients with pancreatic cystic neoplasms (including six mucinous cystadenocarcinomas, two mucinous cystic neoplasms with borderline malignancy, two mucinous cystadenomas, and five serous cystadenomas), 4 patients with pancreatic ductal adenocarcinomas with cystic change, and 10 patients with pseudocysts were studied. Echo-guided or computed tomography-guided biopsies of pancreatic cystic lesions and their normal counterparts were conducted on all patients prior to operation or other management. The specimens were assayed for telomerase activity by using TRAP (telomere repeat amplification protocol). The level of telomerase activity in each specimen was semiquantitated as strong, moderate, weak, and none. The final diagnoses were made from histopathological examination of surgically resected or biopsied specimens. The efficacy of telomerase activity as a tumor marker to predict malignancy of pancreatic cystic lesions was evaluated. RESULTS: Three of the four pancreatic ductal adenocarcinomas with cystic change had strong or moderate telomerase activity; four of the six mucinous cystadenocarcinomas had moderate or weak telomerase activity; one of the two mucinous cystadenomas with borderline malignancy had weak telomerase activity; and none of their normal counterparts had detectable telomerase activity. In contrast, none of the two mucinous cystadenomas, five serous cystadenomas, and 10 pseudocysts had detectable telomerase activity. Based on these results, the sensitivity of telomerase activity for prediction of malignancy or premalignancy of pancreatic cystic lesions was 67%, the specificity was 100%, and the positive and negative predictive values were 1.0 and 0.81, respectively. The overall accuracy was 86%. CONCLUSIONS: The differential expressions of telomerase activity have been detected specifically in malignant and premalignant pancreatic cystic tumors, but not in benign cystic neoplasms or pseudocysts. The implications of these results are that telomerase activation takes part in the malignant transformation of pancreatic cystic neoplasms and that telomerase activity is a useful marker to distinguish malignant pancreatic cystic tumors from benign neoplasms and pseudocysts.

Pancreatic pseudocyst associated with eating disorder.

We describe a patient with an eating disorder and hyperamylasemia originating from the salivary glands, who developed pancreatitis with a huge pancreatic pseudocyst. A 40-year-old woman was referred for the treatment of an eating disorder that had persisted for 9 years. She was admitted with abdominal pain, diarrhea, and nausea. She had bilateral parotid enlargement with marked elevation of total serum amylase level (3288 IU/l; normal range, 60-220) and an isolated increase of salivary isoamylase activity. After her symptoms resolved, oral intake of food was commenced. She subsequently complained of abdominal pain; this was associated with a slight elevation of serum pancreatic isoamylase and lipase levels, and a huge pancreatic pseudocyst was detected. Percutaneous drainage of the pseudocyst was successful. Endoscopic retrograde cholangiopancreatography demonstrated irregularity of the pancreatic duct. Based on these findings, the final diagnosis was parotid enlargement and acute exacerbation of chronic pancreatitis associated with a pancreatic pseudocyst in a patient with an eating disorder.

Pancreatic pseudocyst propagating into retroperitoneum and mediastinum.

The extrahepatic pseudocysts of pancreatic origin sometimes propagate into mediastinum and retroperitoneum. A large pseudocyst of pancreatic origin propagating into the mediastinum up to the aortic arch is published. The surgical intervention has been urged by the dislocation of the heart, by the danger of autodigestion of the mediastinal aorta and by the danger of rupture. Attention is directed to the external drainage operation which is suitable for emptying of the pseudocyst not independently from the ripeness of pseudocyst wall and the characteristics of the pseudocyst content. Should the time short after the exacerbation and should the amylase content high in the cyst, the immediate result of external drainage better and reliable safe.

[Early and late evaluation of Jurasz's operation]. / Wczesna i odlegla ocena operacji Jurasza.

In the years 1971-1996, 102 patients were treated for pancreatic pseudocysts. In 42 cases pancreatocystogastrostomy modo Jurasz was done. 12 patients operated on in the years 1971-83 were included in group I--retrospective trial, other 30 patients treated in the years 1984-96 were included to group II (prospective trial). Postoperative course was uneventful in 38 patients, remaining 4 (9%) had the following complications: bleeding from gastric mucosa 2x, or from the cysts 1 x, mechanical ileus 1x. Hospital mortality was 7% (3 patients). The cause of the death was: sepsis, pulmonary oedema, cardiac arrest. Pancreatic enzymes monitoring in the postoperative period and controlled USG and endoscopic tests (group II) indicate that emptying of the cysts takes place mainly during first 3-4 days and it's closure is completed within two weeks. The follow up period is 1 month to 13 years (mean 5 years). Recurrence of the cyst was noted only in 1 patient 2%.

The proteolytic effect of pancreatic pseudocyst fluid on vessel walls.

The possible in vivo consequences of proteolytic activity found in pancreatic pseudocyst fluid was investigated experimentally using fresh vessels from rabbit and humans. Proteolytic pseudocyst fluid caused a pronounced and time-dependent decrease in breaking strength of fresh vessels. A destruction of the normal histological architecture and fragmentation of the elastic tissue of the vessel wall paralleled the mechanical findings. The proteolytic digestion was caused by the low-mol-wt fraction of the pseudocyst fluid, corresponding to free proteases. Part of this proteolysis was probably caused by pancreatic proteases, since pancreatic juice also caused a decrease in breaking strength and a destruction of the histologic architecture of the vessel wall. Proteases bound to protease inhibitors, i.e., to alpha-2-macroglobulin, had no proteolytic activity. It is concluded that the proteolytic digestion caused by proteolytic pancreatic pseudocyst fluid may well explain severe complications of pancreatic pseudocysts, like bleeding within the pseudocyst and rupture of the entire pseudocyst wall, although the proteolytic digestion is probably counteracted by a constant regeneration of the pseudocyst wall in vivo.

Proteolytic activity in pancreatic pseudocyst fluid.

Pancreatic pseudocyst fluids from 15 patients were biochemically analyzed, especially concerning proteolytic activity and protease inhibitors, and correlated to the clinical course. The pseudocyst fluid was a mixture of pancreatic juice and plasma possessing a high proteolytic activity against high- as well against low-mol-wt proteins. There was practically no functional protease inhibitory capacity left, although immunoreactive inhibitors were present. No distinct biochemical findings differed between fluids from "acute" or from "chronic" pseudocysts. It is concluded, that high proteolytic activity within a pancreatic pseudocyst could well explain symptoms as well as complications caused by the pseudocyst. Biochemical analysis of the pseudocyst fluid cannot, however, be used to differentiate between pseudocysts with a harmless or a complicated course.

[Pancreatic pseudocyst. A review of 10 years]. / Pseudoquiste pancreático. 10 años de revisión.

In the hope of finding those patients whose pancreas pseudocyst resolution were spontaneous, we undertook a retrospective review of such cases admitted at the Hospital Central de San Cristobal y Hospital Patrocinio Peñuela Ruiz (from IVSS), with the diagnosis of acute pancreatitis. From 619 patients with pancreatitis, 40 developed a pseudocyst (5.78%). The most frequent ethiology was biliary disease (47.5%) and abdominal pain in 87.5%, the most common symptom. Abdominal ultrasonography was the best diagnostic aid. Spontaneous resolution occur in 24 cases (60%) in juntion with the normalization of seric and urinary amylases values, the size of the cyst in these patients was less than 5 cms. Sixteen patients needed surgery, in 8 of them the seric amylase value remained high and in 3 cases this value was normal but with a cyst size more than five cms. Internal drainage in 11, external in 4 and surgical resection in one. There was no deaths in this review.

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.

Assessment of value of pancreatic pseudocyst amylase concentration in the treatment of pancreatic pseudocysts by percutaneous evacuation.

The aim of our study was to determine the value of the percutaneous pancreatic pseudocyst evacuation. We assessed the relation between the amylase concentration of the pseudocyst contents and the final outcome of the disease treated by the percutaneous evacuation. Forty-three patients with a history of acute pancreatitis and pancreatic pseudocysts larger than 5 cm in diameter that persisted beyond 6 weeks were divided into four groups relative to the amylase concentration in the pseudocystic contents and the number of evacuations. The results show a good correlation between low amylase concentration in the liquid pseudocystic contents (less than or equal to 64 WU) and the healing rate after the percutaneous evacuation (p less than 0.001). The percutaneous evacuation of the pseudocysts failed in patients with increased amylase concentrations in the pseudocyst fluid regardless of the number of evacuations. We conclude that surgical treatment is indicated in patients who have amylase-rich pseudocyst contents.

Serum pancreatic isoamylase activity in pancreatic disease.

The diagnostic value of serum amylase determination for pancreatic disease has been questioned due to its lack of specificity. Several methods have been developed to separate the tissue-unspecific salivary fractions from the tissue-specific pancreatic fractions. Agarose or cellulose acetate gel electrophoresis are most suitable for clinical practice. The isoamylase patterns were studied by agarose electrophoresis in 55 patients with known pancreatic diseases (acute pancreatitis, pancreatic pseudocysts, exocrine pancreatic insufficiency and pancreatic carcinoma). Increased P-type isoamylase seems to be more sensitive than total amylase in diagnosing acute pancreatitis, while identification of the minor isoamylase P3 is more specific and could have a prognostic value. Detection of low P-type isoamylase levels is an easy method to diagnose exocrine pancreatic insufficiency. Furthermore, a group of patients with pancreatic disease (Pa), was compared with a group of patients with biliary disease without clinical evidence of pancreatic involvement (Bi), and patients with abdominal pain, without evidence of biliary or pancreatic disease (Ab). More than half of the Bi patients presented with abnormal P isoenzyme patterns, whereas 72% of the Ab patients had a normal pattern. Only P3 could distinguish between the Bi and Ab group. This might point to pancreatic involvement in patients presenting with biliary disease, only detected by isoamylase analysis.

Isoamylase determination by isoelectric focusing in pancreatic disorders. A potential clinical aid.

Isoamylase analysis by isoelectric focusing was performed in the serum of 30 healthy volunteers, 65 patients with acute or chronic pancreatic diseases, nine with acute abdomen, four with macroamylasemia, and four with duodenal duplication. In controls, up to four fractions (2 salivary, 2 pancreatic) were found; the pancreatic fractions were as a mean 44.7% (SD 8.6) of total. In chronic pancreatitis, only patients with steatorrhea showed a significant reduction of pancreatic isoamylase (p less than 0.001). In all patients with acute pancreatitis or pseudocysts, an additional fraction (similar to the so-called P3 fraction) was resolved. Moreover, additional isoenzymes were found in all patients with severe acute pancreatitis or pseudocysts, and not in controls or patients with mild forms, acute abdomen or duodenal duplication. A similar pattern was shown in a stored control serum after 10 mo at -20 degrees C. These fractions disappeared after successful surgical drainage. No specific alteration was found in pancreatic cancer. Amylase fractionation by isoelectric focusing can be used to confirm an acute pancreatitis, and to monitor patients with pancreatic pseudocysts and collections after surgical drainage.

[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.

Mucinous cystadenoma: pitfalls of differential diagnosis.

Cystic neoplasms of the pancreas often are difficult to differentiate from pseudocysts. It has been proposed that a history of clinical pancreatitis, elevated serum pancreatic enzymes, elevated cyst fluid amylase, and a communication with the pancreatic duct suggest the diagnosis of a pseudocyst. We report the case of a young woman who presented with a cystic mass in the pancreas and was thought to have a pseudocyst because of the above; at surgery, a mucinous cystadenoma was documented. The pitfalls of differentiating neoplastic cysts of the pancreas from pseudocysts are discussed.

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.

Puzzling persistent hyperamylasemia, probably neither pancreatic nor pathologic.

Increased serum amylase levels most commonly signify pancreatic disease. One hundred seventeen consecutive patients were studied because their serum amylase levels were abnormally high for periods ranging from 3 to 48 weeks. In each case, extensive clinical and radiologic evaluation had failed to reveal a reason for the abnormality. The amylase isoenzymes of their sera were separated by polyacrylamide gel electrophoresis, and the fractions were measured by a saccharogenic assay. The findings in the 117 patients showed that 79 percent had non-pancreatic causes for their hyperamylasemia. The biggest single group (64 percent) had a normal distribution of isoamylases, albeit at unusually high concentrations. This phenomenon, which has not been defined previously, is probably a variant of normal in which the homeostatic balance between production and metabolism is set at a high level. Macroamylasemia accounted for 6 percent of the cases and salivary hyperamylasemia for only 9 percent. Three patients had the characteristic isoamylase pattern ("old amylase") associated with pancreatic pseudocysts. Isoamylase fractionation is a cheap, efficient, and effective means of ruling out a pancreatic cause for hyperamylasemia. It is probable that in the majority of cases of persistent hyperamylasemia without obvious clinical cause there will be no disease at all.

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.

Leukocyte elastase alpha 1-proteinase inhibitor complexes may diagnose pancreatic abscesses early.

Complexes of alpha 1-proteinase inhibitor and leukocyte elastase could be demonstrated by crossed immunoelectrophoresis of the peritoneal fluid from four patients who developed a pancreatic abscess during an attack of pancreatitis. No such complexes were seen in 69 patients with acute pancreatitis without an abscess. The complexes were demonstrable 2-3 days before the abscess was clinically evident. They may thus be diagnostically and therapeutically important. The appearance of these complexes denotes the liberation of large amounts of leukocyte elastase. This may help explain the pathophysiology and high mortality of the pancreatic abscess, since leukocyte elastase is known to cause degradation of all components of connective tissue and also degradation and activation of many components within the different cascade systems.

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.