In spite of improved imaging modalities and emerging clinical awareness of pancreatic cysts, there is currently no assay for use before surgery available with the sensitivity and specificity needed to accurately predict the biological behavior of a pancreatic cyst. In some instances, neoplastic and non-neoplastic cysts can be readily distinguished by imaging, while in other patients this can be considerably challenging. The development of readily applicable guidelines to unequivocally determine which pancreatic cystic lesions can be conservatively managed with regular monitoring and which require surgical resection would be a major advance.36
Pancreatic cystic lesions, both neoplastic and non-neoplastic, are nearly always amenable to aspiration of cystic contents, which provides a unique opportunity for biomarker discovery in biospecimens proximate to lesional tissue.
Cyst fluid can be obtained by EUS-based fine needle aspirate for cytology, but the diagnostic benefit of such aspirations is often hampered by contamination from non-neoplastic tissues (such as the gastric mucosa) as well as by the inability to gather a sufficiently representative sample from a heterogeneously septated cyst. Moreover, EUS-based fine needle aspirate samples from cysts are often lacking adequate cellularity, reducing the chances of obtaining an accurate diagnosis on cytology of the aspirated material. Thus, sampling errors by EUS-based fine needle aspirate might result in false negative diagnosis of an absence of high-grade dysplasia or carcinoma, an outright criterion for resection. Nevertheless, cytologic preparations from fine needle aspirates can aid in the diagnosis of pancreatic cysts. Fine needle aspirates of IPMNs and MCNs cannot usually be distinguished because the ovarian-like stroma of MCNs is usually not present in aspirates. A more general diagnosis of mucin-producing neoplasm, therefore, is often used. Two components predominate in these aspirates: mucin and epithelium. Particularly thick, tenacious, colloid-like mucin is highly suggestive of a mucin-producing cystic lesion. The second component, the epithelium, is needed to establish the diagnosis of a neoplasm. The epithelial cells are typically columnar, and they can form flat sheets or papillary structures. The degree of dysplasia within aspirates often underestimates the degree of dysplasia identified in subsequent resection specimens, presumably because of the considerable heterogeneity that is often present in these lesions.
The diagnostic potential of protein expression patterns in cyst fluid has been extensively investigated. The most promising tumor markers include CEA, CA19-9 and carbohydrate antigen 125 (CA125). Of those, CEA (with a cut off of 200 ng/ml) is most frequently used to distinguish between cysts that produce mucin (IPMNs and MCNs) and nonmucinous cysts. However, a distinction between a noninvasive and an invasive cystic lesion is currently impossible using only the levels of CEA.75–77
Allen et al.
screened cyst fluid specimens by means of a commercially available and custom designed multiplex protein assay using a biomarker panel developed for pancreatic cancer.78
Protein expression profiles were used to discriminate accurately between SCA and IPMN in 92% of patients. Interestingly, the only proteins overexpressed in the cyst fluid of patients with mucin-producing neoplasms were CEA and CA72.4. The majority of the other proteins included in the assay were downregulated in IPMN and MCN fluid specimens, compared to SCA fluid specimens.78
The same group of investigators evaluated the use of cyst fluid CEA analysis in the diagnosis of mucin-producing cysts by correlating clinicopathological and outcome data with CEA levels in cyst fluid collected during EUS. A cut off level of CEA >192 ng/ml proved to be capable of predicting the presence of a mucin-producing cyst with a sensitivity of 73% and a specificity of 65%. However, CEA levels were again not predictive of a malignant mucin-producing cyst or radiographic progression of the lesion.79
By contrast, in a study published in 2010, the value of CEA assessment before surgery for differentiating between a mucin-producing cyst and a nonmucinous cystic lesion has been questioned. The investigators found considerably raised levels of CEA (>450 ng/ml) in 3 of 9 lymphoepithelial cysts, which are benign lesions.80
Levels of amylase in the cyst fluid is another potential parameter in differentiating, for example, a pseudocyst, in which high levels of amylase are usually present, from a cyst that did not arise from pancreatic epithelium, such as a lymphangioma, which usually show low levels of amylase. In addition, SCAs generally have low levels of amylase in their cyst fluid. Since levels of amylase in IPMNs and MCNs are often increased, a differentiation from pseudocysts might not be possible using this analyte alone.81
Thus far, neither CEA nor amylase have been approved by the FDA for biomarker purposes in cyst fluid. These tests, therefore, have to be regarded as off-label applications.1,82
Not surprisingly, there have also been attempts to assess DNA-based markers in cyst fluid as a means of diagnostic stratification. For example, a pilot study used the amount of DNA in cyst fluid and molecular alterations in the DNA to facilitate the underlying diagnosis. A mutation in KRAS
followed by microsatellite loss of heterozygosity at hot spots seemed to be most predictive of the presence of malignancy in a pancreatic cyst.83
mutations and a panel of microsatellite markers were found to correlate with the degree of dysplasia in cysts.84
These initial attempts were expanded into a multicenter trial—the Pancreatic Cyst DNA Analysis Study (PANDA study), which included 113 patients with pancreatic cysts who underwent surgical resection or had diagnostic aspiration cytology.85
The cysts were classified as benign (nonmucinous) or mucinous, with the latter being further subdivided into pre-malignant and malignant (those with carcinoma in situ
or invasive adenocarcinoma, respectively). Cyst fluid DNA was obtained by EUS, and in addition to analysis of mutations in KRAS
, the overall fraction of alleles deemed as lost compared to the germ line (mean allelic loss amplitude or MALA) was determined.
In the PANDA study, the presence of a mutant KRAS
or an MALA >65% were predictive of a mucinous lesion by both univariate and multivariate analysis, and DNA analysis improved the sensitivity of cyst fluid CEA. Notably mutation of KRAS
was by itself not predictive of malignancy in mucin-producing cysts (probably a reflection of these mutations occurring early), although an MALA >82%, as well as the combination of KRAS
and high MALA were associated with high-grade cysts that produced mucin. Although the PANDA study provides some rationale for coopting DNA-based analyses in diagnosis of pancreatic cysts, there were inherent weaknesses in the study design that diminish the overall significance of the results.86,87
For example, the study cohort had a selection bias towards high-grade cysts than are typically observed in a nonselected population; additionally, it was unclear from the trial if DNA analysis (an expensive addition to health-care costs) would add value to optimally applied Sendai criteria alone. Finally, there is concern that allelic loss amplitude can be confounded by DNA degradation as well as a number of variables attributable to underlying heterogeneity in cyst fluid composition. One anticipates that genomic profiling of IPMNs and MCNs by next-generation technologies will elucidate improved and cost-effective diagnostic biomarkers that can fill the current void in this area.
In addition to genomic alterations, epigenetic abnormalities, such as DNA methylation and microRNA (miRNA) expression profiles, have also been examined as potential biomarkers in cyst fluid samples.88,89
miRNAs are noncoding RNAs, ~21–23 nucleotides in length, that are involved in transcriptional regulation of coding genes.90
Aberrant miRNA expression has emerged as one of the hallmarks of human neoplasia, including pancreatic cancer.91–93
Our group assessed the relative expression levels of a panel of 12 miRNAs known to be upregulated in PDAC in 15 noninvasive IPMNs. miR-21 (mean 12.1-fold increase) and miR-155 (mean 11.6-fold increase) were identified as the most promising biomarker candidates. Furthermore, upregulation of miR-155 transcripts were observed in 6 of 10 IPMN-associated pancreatic fluid specimens compared with none of the specimens from control individuals.88
Analysis of the levels of miRNA expression in cyst fluid samples, therefore, might serve as an important tool in the diagnostic and prognostic classification of pancreatic cysts. This approach deserves further investigation.