pancreatic cysts are increasing in prevalence as cross - sectional imaging has become widely utilized .   in recent population - based studies using magnetic resonance imaging ( mri ) [ 14 ] and computerized tomography ( ct ) scans     [ 5 , 6 ] , the estimated prevalence of cystic lesions ranges from 2.6% to as high as 44.7% . not surprisingly , the number of evaluations for these lesions is increasing     [ 8 , 9 ] . management of these increasingly prevalent lesions can utilize a significant amount of health care resources in the form of diagnostic studies and surgical resections . therefore , developing an accurate and cost - effective diagnostic test to assist in patient management is a clear priority . this paper will focus on cystic neoplasms which are distinguished by the presence of mucinous or nonmucinous epithelium . ninety percent of all cystic neoplasms are comprised of serous cystadenomas ( scas ) , a non - mucinous lesion , as well as mucin - producing cystic tumors comprised of mucinous cystic neoplasms ( mcns ) and intraductal papillary mucinous neoplasms ( ipmns )     . rare cystic neoplasms include solid pseudopapillary lesions , lymphoepithelial cysts , and cystic degeneration of pancreatic ductal adenocarcinoma or neuroendocrine tumors ( see table 1 ) . the most common nonneoplastic cyst is a pancreatic pseudocyst which is associated with acute pancreatitis and has no epithelium . they tend to occur predominantly in women ( 87% ) with a median age in the early 50s     [ 11 , 12 ] . scas are usually comprised of microcystic components , with a classic honeycomb appearance , though they can be macrocystic in appearance . up to 30% of these lesions will have a characteristic central scar . their malignant potential is considered so low that they are generally not resected unless symptomatic . mcns have columnar mucinous epithelium with surrounding ovarian stroma ( defined as hypercellular spindle cell bundles that lay just beneath the epithelium and usually show positive staining for estrogen and progesterone receptors )     [ 13 , 14 ] and typically present as large solitary macrocystic lesions in the body or tail of the pancreas . they are thought to be separate from the main pancreatic duct but can be connected in up to 20% of cases     . they occur almost exclusively in women ( 9598% )      during the fourth or fifth decades of life . the rate of malignancy ranges from 630% at the time of resection     [ 1517 ] . risk factors for malignancy include older age , the presence of a mural nodule with the cyst , and cyst size > 4  cm     [ 14 , 15 , 17 ] .    the five - year survival is 100% in patients with benign disease and 60% in those patients that develop invasive carcinoma [ 17 , 18 ] . ipmns are characterized by columnar papillary mucinous epithelium that involves the main pancreatic duct , the side branch ducts ( sb - ipmn ) or both ( mixed ipmn ) . ipmns tend to occur more frequently in the head of the pancreas than the body and tail . the risk of malignancy is much higher in main duct disease ( mean 70% ) than side - branch disease ( mean 25% ) .    the reported overall 5-year survival rate for resected noninvasive ipmn ranges from 77 to 100% , whereas 5-year survival rate for invasive ipmn ranges from 30% to 75%     . recently , ipmns have been categorized into four epithelial subtypes  gastric , intestinal , pancreaticobiliary , and oncocytic , based upon cell morphology and expression patterns of glycoproteins containing mucin ( muc )     . combinations of epithelium subtypes may be present within an individual lesion and therefore each ipmn is classified by the dominant component     .   based upon recent studies , these categories may explain the clinical behavior of the different ipmn subtypes     [ 23 , 24 ] . gastric - type ipmns primarily are located in the side branches and express muc5ac but not muc1 or muc2     . these rarely undergo malignant transformation     , but if they do , they develop into tubular adenocarcinomas , which have a survival that is almost as poor as ductal adenocarcinoma . intestinal - type ipmns are located mainly in the main pancreatic duct and express both muc2 and muc5ac     . these have a high frequency of malignant transformation to colloid carcinoma , which has a better prognosis than ductal adenocarcinoma     [ 23 , 24 ] . when compared to the other subtypes of ipmn , pancreaticobiliary - type ipmn is noted to occur more frequently in women than men and at a later age ( mean 69.2  y versus 60.365.6  y )     .   by immunohistochemistry , they express muc1 and muc5ac     [ 22 , 23 ] and may progress to tubular adenocarcinoma     [ 23 , 24 ] . these tend to develop in people at a younger age than the other ipmn subtypes     . while these can progress to malignancy ( oncocytic adenocarcinoma ) , they tend to be noninvasive      and have better survival than ductal adenocarcinoma     [ 23 , 24 ] .     to date , the subtypes of mucinous tumors ( mcn and ipmn ) can only reliably be distinguished by surgical pathology . there are no presurgical tests that distinguish these cyst types with a high level of accuracy . moreover , the triggers or markers for malignant transformation are unknown and the timeline to transformation remains unclear .   as such , our knowledge of the natural history of these lesions is still limited . in 2006 , international consensus guidelines were developed by a team of experts to define management of cystic mucinous neoplasms .   for cystic neoplasms , the decision to undergo surgical resection versus surveillance should be tempered by patient 's wishes , comorbidities , life expectancy , and the risk of malignancy versus the risk of surgery . if the patient is an appropriate surgical candidate , the guidelines suggest resection of all mcns and any ipmn which involve the main duct , or side branch ipmn lesions that are symptomatic , have a solid component , or are > 3  cm in size . they recommend yearly surveillance for lesions < 10  mm , and surveillance every 612 months for lesions 1020  mm and every 36 months for lesions > 20  mm . a retrospective study of 147 patients demonstrated that the algorithm proposed by the guidelines had a sensitivity and negative predictive value of 100% but a specificity of 23% . the algorithm therefore seems reasonably sensitive to identify those who do not need surgery , but given the low specificity , there remains a fairly high resection rate for patients with benign disease     . the clinical challenges of managing patients with pancreatic cystic neoplasms have several layers of complexity . non - mucinous lesions generally do not require follow - up , whereas because of their premalignant potential , mucinous neoplasms are either resected or monitored in a surveillance program . second , once a mucinous lesion is identified , one should distinguish between mcn and ipmn ( in particular focal sb - ipmn ) since the former should be resected whereas the latter can be monitored . this can be a difficult task in part because mcns occasionally have communication with the main duct which is sometimes difficult to accurately identify by cross - sectional imaging or eus     [ 10 , 20 ] . moreover , there is no preoperative test that can identify the characteristic ovarian stroma of mcns     , and even on surgical pathologic analysis , the stroma may not be uniformly present , particularly with malignant transformation . third , amongst the mucinous lesions , one must differentiate between those that have high - grade dysplasia and cancer and those that are benign in order to appropriately refer those patients to surgery who would most benefit from resection . this differentiation would allow more selective recommendation of surgical resection for those who truly need it . finally , given that the natural history of these lesions remains to be clarified , one must be able to identify those lesions that will go onto malignant transformation .   to date , no single test or tests adequately addresses these challenges and as such , a biomarker or set of biomarkers are needed in order to address all four of these challenges . current tests have limited ability to distinguish between mucinous and non - mucinous lesions or to identify malignant cysts ( figure 1 ) . cross - sectional radiologic imaging is limited in its ability to distinguish between the different types of cysts . the accuracy of ct and mri to determine the correct histology ranges from 4060%     [ 32 , 33 ] . new advances in ct and mri technology that provide more detailed images may account for a modest increase in accuracy up to 84%     . morphology by endoscopic ultrasound is also limited in its ability to distinguish between types of cystic tumors , with a sensitivity and specificity of 56% and 45% , respectively . furthermore , the accuracy of eus morphology is limited by a lack of interobserver agreement     . the tests of choice for diagnostic evaluation include cytology and carcinoembryonic antigen ( cea ) . fluid cytology can be limited due to luminal contamination , highly variable amounts of extracellular mucin , and scant cellularity within the cyst . the overall accuracy of cytology in identifying mucinous lesions is around 50%     [ 35 , 38 ] . there are no cytological findings which currently distinguish mcn from ipmn     [ 39 , 40 ] . cytology has a specificity that approaches 100% but lower sensitivity in identifying the presence of malignancy     [ 38 , 41 , 42 ] . to date , cyst fluid cea remains the most accurate test to distinguish mucinous from non - mucinous cysts . a prospective , multicenter study determined that a cea > 192  ng / ml had a 75% sensitivity and 85% specificity in distinguishing between mucinous and non - mucinous cysts . its overall accuracy of 79% was higher than morphology , cytology , and other tumor markers previously identified in pancreatic cysts such as ca72 - 4 , ca19 - 9 , and   ca 15 - 3     [ 35 , 45 ] . however , fluid cea is limited by the fact that there is broad overlap between types of lesions . in addition , this test is unable to distinguish between types of mucinous cysts , nor is there any correlation between elevated concentrations and risk of malignancy . as a result of these current limitations , there has been considerable interest in finding other biomarkers that can better distinguish mucinous lesions and identify patients with tumors of higher malignant potential ( i.e. , with high - grade dysplasia or carcinoma ) who would benefit most from surgical resection . the general approach that has been taken is to aspirate cyst fluid and use a variety of techniques to try   to create highly sensitive and specific assays to identify subjects with high grade dysplasia or frank malignancy . pancreatic cyst fluid would appear to be an ideal source for a biomarker development due to its relative ease at obtainment by endoscopic ultrasound , which has   a low complication rate when performed by an experienced endoscopist , and the presumed localization of relevant biological material from cyst epithelium . there is a considerable interest in genetic material within cyst fluid and its potential as to serve as biomarkers . dna mutations , such as k - ras , and allelic loss amplitude of a proprietary list of specific pancreatic cancer - related genes within cyst fluid have been studied as surrogate markers for mucinous and malignant cysts     . in a multicenter study of 113 patients , the authors reported that the presence of a cyst fluid k - ras mutation had a high specificity of 95% but low sensitivity of 45% for diagnosing mucinous cysts . the combination of a k - ras mutation followed by allelic loss showed a high specificity of 96% but a low sensitivity of 37% in diagnosing malignant cysts     . subsequent studies have reported mixed correlation between these dna mutations and final surgical pathology     [ 5052 ] . the added benefit over existing tests remains unclear , and as such , the role for dna analysis will need to be clarified     [ 46 , 53 ] . micrornas ( mirnas ) are small ( 22 nucleotides ) noncoding rnas that regulate the stability and translation of mrna transcripts . deregulation of mirna expression has been identified in several human cancers , including pancreatic adenocarcinoma     [ 5459 ] . using a panel of 12 mirnas that are upregulated in pancreatic cancer , habbe et al . described the identification of abnormal mirna expression in   surgical histology from 15 noninvasive ipmns compared to normal pancreatic tissue . moreover , they established the feasibility of identifying mirna in pancreatic juice .   the two mirnas with the highest expression , mir-21 and mir-155 , both of which have been shown in laboratory studies to inhibit apoptosis , had higher expression in the ipmns ( 6 of 10 , 60% ) than normal controls ( 0 of 5 ) , though this did not reach statistical significance due to a small number of samples . there was also an increased frequency of mir-155 expression in ipmn lesions with pancreaticobiliary and intestinal epithelium . further studies will be required to validate these findings and to define the true utility of using mirnas as biomarkers in pancreatic cyst fluid . another strategy for biomarker development is to identify specific proteins already known to be involved in pancreatic cancer . one group used multiplex assays with 54 proteins associated with pancreatic cancer to demonstrate differential protein expression between noninvasive ipmn and sca ( 34 out of 51 proteins , 67% ) and noninvasive mcn and sca ( 13 out of 51 proteins , 25% )     .   when using a panel of 14 proteins , the accuracy of distinguishing ipmns from scas was 92%     . another group took a more specific approach and examined the role of prostaglandin e(2 ) , which they had shown to have increased expression in pancreatic cancer tissue over normal pancreatic tissue     , in distinguishing between types of mucinous cysts .   from fluid obtained from 58 resected cystic lesions , they demonstrated that cyst fluid pge(2 ) concentrations were greater in ipmns versus mcns ( 2.2  0.6 versus 0.2  0.1  pg / mol , p < 0.05 ) and that the mean level of pge(2 ) increased with the degree of dysplasia in ipmn lesions . however , there was noted to be an overlap in pge(2 ) concentrations in benign mcns ( n = 11 ) and scas ( 9n = 5 ) , thereby limiting the utility of this biomarker in the clinical setting . these studies demonstrate that targeting proteins associated with pancreatic cancer show potential in identifying appropriate biomarkers for cystic lesions and will require further investigation and validation . as described above , immunohistochemistry of surgical pathology has demonstrated differential mucin profiles in ipmn     [ 64 , 65 ] and it would seem logical to expect mucin profiles in cyst fluid to identify lesions at risk for malignancy . a recent study has demonstrated differential mucin expression in cyst fluid from 40 surgically resected ipmns using enzyme - linked immunosorbent assays     . patients with high grade dysplasia or carcinoma ( n = 19 ) were categorized as  high risk . cyst fluids muc2 and muc4 were elevated in high - risk patients as compared to low risk patients ( 10  3.0  ng / ml versus 4.4  1.2  ng / ml , p = 0.03 ; 20.06  10.6  ng / ml versus 4.5    1.4  ng / ml , p = 0.03 , resp . ) . cysts with gastric epithelium ( n = 23 ) had statistically significant lower expressions of muc2 , muc4 , and muc5ac compared to pancreatic cystic tumors without gastric epithelium . cysts with intestinal epithelium ( n = 8) had statistically significant higher elevations of muc2 compared to nonintestinal cysts and a trend towards higher muc4 concentrations . there was no discernible difference in muc concentrations in pancreaticobiliary epithelium cysts compared to those without pancreaticobiliary epithelium . these findings have not yet been validated ; however , this study highlights the potential for risk stratification based upon muc expression in cyst fluid . other proteins previously identified in pancreatic cancer have also been studied to more accurately identify ipmn harboring malignancy . mutant k - ras protein has been identified in cyst fluid using mass spectrometry . the expression of plectin-1 , a marker found to be increased in ductal adenocarcinoma , has also been identified in fluid from malignant mucinous cysts . cytokine il-1 is   markedly elevated in high - risk patients compared to low risk patients in a small number of ipmns . proteomics is an attractive method for identifying proteins within the cyst fluid which can differentiate mucinous cysts or identify malignancy with greater accuracy . traditionally , proteins are separated by two - dimensional gel electrophoresis with subsequent mass spectrometric identification of protein spots or by protein digestion and mass spectrometric identification of peptide sequences . proteomics overcomes the shortcoming of using dna or mrna analysis , whose changes may not reflect actual protein expression     [ 70 , 71 ] or include posttranslational modifications . the method can identify a large number of proteins but interpretation of the results may be clouded by the signal of the most abundant proteins , and thus proteins present in very small concentrations may not be easy to identify .   within the pancreatic cyst fluid itself , the vast variety of proteins are a reflection of cyst epithelium , luminal contamination ( if fluid is obtained by transgastric or transduodenal aspiration ) , plasma proteins , mucus , and possibly pancreatic enzymes , if there is a connection of the cyst to the pancreatic ductal system .   protein concentration yield may be subject to degradation by endogenous peptidases   or post - translational modifications . proteomics has been used to successfully identify potential biomarkers in the tissue     [ 74 , 75 ] , serum     , and pancreatic juice     [ 77 , 78 ] of patients with pancreatic ductal adenocarcinoma .   a method to perform proteomic analysis using paraffin - embedded archival slides of a noninvasive ipmn carcinoma - in - situ has also been described . the feasibility of proteomic analysis of pancreatic cyst fluid was established by scarlett et al . . in this proof of concept study , cyst fluids from 10 patients ( including 3 ductal adenocarcinomas , 2 mucinous cystadenoma , and 1 ipmn ) were analyzed using seldi - tof mass spectrometry . reproducible protein profiles were demonstrated amongst the adenocarcinoma patients with differential expression in twelve protein peaks identified . these findings suggest that proteomics is a viable method for identifying potential biomarkers within cyst fluid . two recent studies advanced the use of proteomic analysis to identify biomarkers in cyst fluid . ke et al . used small volumes ( < 40  l ) from eus fine needle aspirates and grouped patients according to their cytology results ( ( a ) benign : no evidence of benign mucinous epithelium , atypical cells or carcinoma ; ( b ) benign mucinous epithelium ; ( c ) atypical / suspicious ; ( d ) malignant )     . fluid was analyzed using maldi - tof mass spectrometry with lc / ms / ms protein identification , 2d gel electrophoresis , or gelc / ms / ms ( tryptic digestion of proteins fractionated by sds - page and identified by lc / ms / ms ) .   the first two techniques proved to be unsatisfactory , presumably from endogenous peptidases which splintered native proteins in numerous locations     . mass spectrometry yielded homologs within three families of proteins associated with pancreatic cancer , including mucins , ceacams , and s100s .   the authors conclude that lc / ms / ms mass spectrometry provides useful information on biomarkers within cyst fluid using small volumes of fluid . the same technique was used by cuoghi et al . in a study of 8 patients who underwent surgical resection for symptomatic pancreatic neoplasms . fluid was aspirated directly from the surgical specimens , thereby avoiding potential gastrointestinal luminal contamination . proteins were separated by sds - page and then analyzed by lc / ms / ms .   the total number of proteins identified in the cyst samples ranged from 220 to 727 . they identified 38 proteins unique to neuroendocrine tumors , 18 unique to serous cystadenomas , 92 unique to mcns , and 29 unique to ipmns . analysis of known proteins revealed that several proteins identified in the mucinous lesions ( mcns and ipmns ) were previously reported to be upregulated pancreatic cancer - associated proteins . the findings were confirmed by immunohistochemistry for two of the identified proteins , olfactomedin-4 ( olfm4 ) and the cell surface glycoprotein muc18 . clearly , proteomics shows great promise in identifying potential biomarkers ( see table 2 ) . further studies and refinement of technique will hopefully yield reliable candidate biomarkers that can be validated in clinical studies . glycoproteomics specifically examines carbohydrate modification or glycosylation of proteins .    aberrant glycosylation is a hallmark for tumorigenesis and tumor progression and the advantage of glycoproteomics is the focused isolation of glycoproteins by specific binding of glycosylation sites . analytic approaches have been broadly categorized as glycoprotein - based analysis or glycopeptide - based analysis     [ 84 , 85 ] . the former begins with enrichment of glycoproteins using lectin and separation techniques to enrich the protein fractions . a technique using lectin affinity chromatography , liquid separation , and characterization by mass spectrometry was demonstrated in serum of patients with pancreatic cancer . ninety - two individual glycosylation sites with 41 glycoproteins were identified and 202 glycan peaks with 104 unique carbohydrate structures were detected during glycan profiling using different separation techniques . forty - five oligosaccharides were found altered in pancreatic cancer serum of which 44 were distinct in the cancer sample .   based on these promising results , glycoprotein microarrays have been created as a high throughput tool to differentiate serum samples from patients with pancreatic cancer , from chronic pancreatitis and normal subjects     [ 86 , 87 ] . this approach has been expanded to the use of glycoproteomics in cystic neoplasms .   using a novel antibody - lectin sandwich array ( alsa ) that targets glycan moieties on proteins ,   haab et al . measured protein expression and glycosylation of muc1 , muc5ac , muc16 , cea , and other proteins associated with pancreatic cancer in 53 cyst fluid samples from surgically resected lesions ( 17 mcn , 15 ipmn , 15 sca , and 9 pseudocysts )     . wheat germ agglutination of muc5ac was markedly elevated in mcn and ipmn but not scas or pseudocysts . ca19 - 9 could distinguish between mcn and ipmn with a sensitivity and specificity of 82% and 93% , respectively . muc5ac in combination with ca19 - 9 ( sensitivity 87% , specificity 86% ) outperformed fluid cea ( 37% sensitivity , 80% specificity ) in distinguishing mucinous from nonmucinous cysts . this study shows that glycan variants of proteins within cyst fluid may prove to be useful biomarkers and highlights an area warranting further evaluation . , it remains to be determined if this approach will be useful in separating malignant and non - malignant lesions . the clinical management of pancreatic cystic neoplasms is difficult due to the lack of sufficiently sensitive and specific diagnostic tests to differentiate cyst types and the presence of malignancy . pancreatic cyst fluid provides an appealing source for improved biomarker development , particularly by proteomic analysis . preliminary work with cyst fluid glycosylated mucins show promise in distinguishing mucinous from non - mucinous cysts and differentiating types of mucinous cysts . cyst fluid homologs of mucin , ceacams and s100s , and other proteins associated with pancreatic tumorigenesis have been identified as potential biomarkers for malignancy within cyst fluid . these results will all need to be studied and validated in larger more adequately sized test and training sets of pancreatic cyst fluid for full biomarker development . given that the field is currently limited by the lack of adequate numbers of pancreatic cyst fluid samples for analysis , it will be important that resources for fluid samples are further developed . as we close our gaps in knowledge regarding natural history of mucinous cysts and the relationship between epithelial subtypes and prognosis , biomarkers will likely play a prominent role in the management of cystic neoplasms .
proteomics is a powerful method used to identify , characterize , and quantify proteins within biologic samples . 
 pancreatic cystic neoplasms are a common clinical entity and represent a diagnostic and management challenge due to difficulties in accurately diagnosing cystic lesions with malignant potential and assessing the risk of malignant degeneration . 
 currently , cytology and other biomarkers in cyst fluid have had limited success in accurately distinguishing both the type of cystic neoplasm and the presence of malignancy . 
 emerging data suggests that the use of protein - based biomarkers may have greater utility in helping clinicians correctly diagnose the type of cyst and to identify which cystic neoplasms are malignant . 
 several candidate proteins have been identified within pancreatic cystic neoplasms as potential biomarkers . 
 future studies will be needed to validate these findings and move these biomarkers into the clinical setting .