Many pancreatic cystic lesions have malignant potential, including branch duct and mixed intraductal papillary mucinous neoplasms (BD-IPMNs). As the malignant risk is substantially greater for mixed IPMN than BD-IPMN, current management of mixed IPMN is surgical, while many BD-IPMN may be managed conservatively. Therefore, accurately distinguishing them has important clinical implications. Diagnosis of these lesions relies mainly on the combination of diagnostic imaging and analysis of cyst fluid obtained during endoscopic ultrasound-guided fine needle aspiration (EUS-FNA). While EUS-FNA is safe [
Differentially expressed inflammatory mediator proteins (IMPs) may serve as diagnostic biomarkers for pancreatic cystic neoplasms. IMPs, which include cytokines, chemokines, and growth factors, are commonly associated with acute and chronic disease states. Cytokines are low molecular weight regulatory proteins produced by various cell types particularly during cellular stress events. Generally released in picomolar amounts, their concentration can increase over 1000-fold during physiological stress, such as trauma or infection [
The simultaneous analysis of numerous IMPs can be performed in a single experiment with a suspension microarray using IMP-specific capture antibodies coupled to color-coded microspheres. Current IMP microarrays are both sensitive to low concentrations of cytokines and amenable to high-throughput analysis [
The primary objective of our exploratory investigation is to compare IMP profiles in endoscopically collected pancreatic cyst fluid aspirates of known BD-IPMN and mixed IPMN using a multiplexed IMP-targeted microarray.
The study was designed to analyze IMPs in endoscopically collected pancreatic cyst fluid using a multiplexed suspension microarray assay in an academic center. This protocol was approved by the Partners Institutional Review Board. The study population included adult patients referred to the Center for Pancreatic Diseases at Brigham and Women’s Hospital for evaluation of pancreatic cystic lesions. All subjects underwent the following: (1) comprehensive history and physical examination, (2) review of radiologic data, and (3) EUS-FNA and/or endoscopic retrograde cholangiopancreatography (ERCP).
Only patients with diagnoses of BD-IPMN and mixed IPMN were included. Definitive diagnosis was obtained from a combination of methodologies: a physician review of the patient medical history with radiologic imaging, endoscopic findings, and/or surgical pathology. A single abdominal radiologist (NS), blinded to the official radiology report, reviewed the radiologic studies, which included both abdominal computed tomography (CT) and magnetic resonance cholangiopancreatography (MRCP). By radiology and/or EUS, BD-IPMN was defined as a unilocular or multiloculated pancreatic cyst with smooth or loculated margins with a demonstrable communication (short neck or long channel) to a nondilated main pancreatic duct [
Imaging of BD-IPMN and mixed IPMN. (a) MRI of BD-IPMN: arrow points to communication between BD-IPMN and normal main pancreatic duct. (b) EUS of BD-IPMN: arrow points to communication between cyst and main pancreatic duct. (c) MRI of mixed IPMN: arrow points to diffusely massively dilated main pancreatic duct.
The overall analysis proceeded as follows: (A) EUS-FNA or ERCP sample collection, (B) particulate removal via centrifugation, (C) multiplexed IMP microarray assays, and (D) statistical analysis of the resulting profiles.
Endosonography was performed with a curvilinear echoendoscope (Olympus GF-UC(T)140P-OL5; Olympus America Inc., Center Valley, PA) using Aloka SSD-Alpha 5 and Alpha 10 (Olympus America Inc., Center Valley, PA) processors. Curvilinear echoendoscopes are modified, oblique forward-viewing instruments with curved linear ultrasound transducers providing real-time visualization of the aspiration needle. In brief, after obtaining informed consent and administration of intravenous conscious sedation, the echoendoscope was advanced into the upper gastrointestinal tract, the target lesion located, and FNA of the cystic lesion performed using a 22-gauge adjustable needle (EZ Shot, Olympus, Center Valley, PA). Aspirates were divided into three aliquots for (1) biochemical analysis of CEA and amylase, (2) IMP assay, and (3) cytologic evaluation with fluid placed into Cytolyt preservative (Cytyc, Boxborough, MA). Samples were stored at −80°C until IMP analysis (see Section
The ERCP procedure proceeded in a similar manner to EUS with the exception of using a duodenoscope (Olympus TJF-160VF; Olympus America Inc., Center Valley, PA) and cannula (Tandem XL M00535700; Boston Scientific, Natick, MA) to cannulate the pancreatic duct. Pancreatic duct fluid was aspirated through the cannula and samples sent for analyses.
A suspension microarray assay was used to measure the concentration of 89 IMPs in pancreatic cyst fluid samples from 10 individuals. We selected this 89-cytokine panel as it was the most comprehensive panel available at the time of this study. A list of the IMPs investigated with their corresponding abbreviations is provided in Supplementary Table 1 available online at doi:10.1155/2012/247309. Unlike mass spectrometry-based proteomic assays of pancreatic fluids [
Immediately prior to the microarray analysis, the concentration of known standards was determined by a 5-parameter logistic regression algorithm with analysis of the median fluorescence intensity readings on an 8-point protein standard curve. This procedure ensured that the reading was within the linear range of the assay. The fluorescence intensity values of the standards were treated as unknowns, and the concentration of each standard was calculated using the derived regression equation. The ratio of the calculated value to the expected value of this standard was determined. A ratio between 70 and 130% for each standard indicated a good fit. If fluorescence intensity values of samples plateaued or were outside the range of standard curves, a retest with diluted samples was performed to ensure that the fluorescence intensity measurement of unknown samples fell inside the linear range of standard curves.
Levels of IMPs in cyst fluid were determined using microsphere-based suspension microarray technology (AssayGate, Ijamsville, MD) [
IMP concentrations were expressed in picograms per milliliter (pg/mL) of pancreatic cyst fluid and analyzed by the Kruskal-Wallis one-way analysis of variance by a rank test for two samples using SAS 9.2 (Cary, NC). A
The demographics and clinical characteristics of the 10 study subjects are shown in Table
Patient characteristics.
Patient | Age (yr) | Gender | CEA (ng/mL) | Amylase |
Cytology | Imaging | Endoscopy | Surgical pathology |
---|---|---|---|---|---|---|---|---|
B1 | 81 | Female | 87.4 | 14700 | EUS-FNA: no malignant cells | MRI: multiple septated cysts, largest 19 mm communicating with main PD 6 mm | EUS: multiple cysts, 17 mm communicating with main PD | BD-IPMN, moderate dysplasia |
B2 | 61 | Female | 8.8 | 3 | EUS-FNA: no malignant cells | MRI: 23 mm cyst communicating with nondilated main PD | EUS: 22 mm pancreatic cyst with nondilated 2 mm main PD | N/A |
B3 | 60 | Female | 5980 | 40085 | EUS-FNA: nondiagnostic | CT: 19 mm cyst communicating with nondilated main PD | EUS: 21 mm septated pancreatic cyst | BD-IPMN, LGD |
B4 | 69 | Male | 2363 | 86.7 | EUS-FNA: nondiagnostic | MRI: 19 mm septated cyst not communicating with nondilated main PD | EUS: 14 mm pancreatic cyst | BD-IPMN, LGD |
B5 | 70 | Female | 2782 | 65 | EUS-FNA: nondiagnostic | MRI: multiple septated cyst, largest 19 mm communicating with nondilated main PD | EUS: 13 mm pancreatic cyst with nondilated 1.6 mm main PD | N/A |
M1 | 67 | Female | 632.2 | 37574 | EUS-FNA of cysts in tail: atypical cells | MRI: multiple BD-IPMN with main PD dilation consistent with mixed IPMN | EUS: 3.2 × 1.8 cm cluster of cysts in tail | Mixed IPMN, HGD |
M2 | 86 | Female | N/A | N/A | ERCP: IPMN, LGD | MRI: diffusely dilated main PD, 5.2 cm cyst head/uncinate pancreas consistent with mixed IPMN | ERCP: mucus at papilla | Mixed IPMN, LGD |
M3 | 85 | Female | N/A | N/A | ERCP: adenocarcinoma | CT: diffuse MD-IPMN | EUS and ERCP: massively dilated main PD, mucus at papilla | Mixed IPMN, HGD, foci |
M4 | 78 | Female | 46 | <10 | EUS-FNA of cyst in body: |
CT: 2.4 cm cyst in body of pancreas | EUS: 3.3 cm cyst in body | Mixed IPMN, LGD |
M5 | 79 | Male | 20 | N/A | EUS-FNA of cyst in uncinate: |
CT and MRI: mixed IPMN with 4.8 cm cyst in head of pancreas and dilated main PD | EUS: 3.4 cm cyst in uncinate with dilated main PD 7 mm | N/A |
N/A: not available; LGD: low grade dysplasia; HGD: high grade dysplasia.
The concentration of IMPs ranged from below the limit of detection to greater than 15,000 pg/mL, and several IMPs had median concentrations above >1000 pg/mL. In the BD-IPMN samples, ENA-78 and NAP2 were detected with median concentrations greater than 1000 pg/mL. Similarly in the mixed IPMN samples, HCC1, ICAM1, MIF, NAP2, PDGF-AA, and SCGF-B had median concentrations greater than 1000 pg/mL. Figure
Venn diagram of IMPs identified in BD-IPMN and mixed IPMN. IMPs detected only in mixed IPMN are listed to the right of the diagram. Of the 89 IMPs assayed, three were not detected in either types of cyst (b-NGF, IL-11 and IL-29).
Inflammatory mediator proteins (
Cytokine | Mixed IPMN | ||||||
---|---|---|---|---|---|---|---|
Concentration, pg/mL | |||||||
Samples | Median | IQR | |||||
M1 | M2 | M3 | M4 | M5 | |||
Eotaxin-3 | N.D. | 91.2 | 11.8 | N.D. | N.D. | 51.5 | 39.7 |
GM-CSF | N.D. | 127.5 | 9.3 | N.D. | N.D. | 68.4 | 59.1 |
I-309 | N.D. | 2.4 | 2.0 | N.D. | N.D. | 2.2 | 0.2 |
IL-5 | N.D. | 11.7 | 0.5 | N.D. | N.D. | 6.1 | 5.6 |
IL-9 | N.D. | 14.2 | N.D. | N.D. | N.D. | 14.2 | 0.0 |
IL-17 | N.D. | 15.7 | 6.7 | N.D. | N.D. | 11.2 | 4.5 |
Lymphotactin | N.D. | N.D. | N.D. | N.D. | 37.2 | 37.2 | 0.0 |
SCF | N.D. | 6.4 | 2.3 | N.D. | N.D. | 4.3 | 2.1 |
TGF- |
N.D. | 104.3 | 94.5 | N.D. | 81.2 | 94.5 | 11.5 |
TGF- |
N.D. | 170.7 | 41.4 | N.D. | N.D. | 106.0 | 64.7 |
TGF- |
N.D. | 14.7 | N.D. | N.D. | N.D. | 14.7 | 0.0 |
TNF- |
N.D. | 3.8 | 8.6 | N.D. | N.D. | 6.2 | 2.4 |
TPO | N.D. | 76.7 | 26.7 | N.D. | N.D. | 51.7 | 25.0 |
TSLP | N.D. | 6.3 | N.D. | N.D. | N.D. | 6.3 | 0.0 |
IQR: interquartile range; N.D.: not detected.
The following IMPs were all present in mixed IPMN and not detected in BD-IPMN samples: eotaxin 3, GM-CSF, I-309, IL-5, IL-9, IL-17, lymphotactin, TGF-
Box and whisker plots of differentially expressed IMPs between BD-IPMN and mixed IPMN. (a) G-CSF,
Among the 72 IMPs detected in both BD-IPMN and mixed IPMN samples, G-CSF (
We identified IMPs with a microsphere-based suspension protein microarray assay in all endoscopically obtained pancreatic cyst fluid samples from patients with BD-IPMN and mixed IPMN. Our study differentiated the IMP profiles of BD-IPMN and mixed IPMN fluid aspirates. Specifically, we identified a total of 17 IMPs from the 89 tested that were differentially expressed between BD-IPMN and mixed IPMN. Fourteen IMPs were detected only in mixed IPMN, while three IMPs were present in higher concentrations in mixed IPMN.
Accurate differentiation between BD-IPMN and mixed IPMN has important clinical implications. Mixed IPMN harbors a risk of malignancy up to 50–70%, similar to main duct IPMN (MD-IPMN), compared to approximately 15–25% for BD-IPMN; therefore, current guidelines recommend surgical resection of mixed IPMN [
Cytokine and chemokine production is closely linked to pancreatic stellate cell function, particularly in the pathogenesis of pancreatic cancer [
The TGF-
G-CSF, IL-23, and VCAM-1 had higher expression levels in mixed IPMN compared to BD-IPMN and also represent potential diagnostic biomarkers. Additionally, these cytokines may lead to insights into the oncogenic nature of these pancreatic cystic neoplasms. Pancreatic cancer has been associated with elevated serum G-CSF [
Our results demonstrate the applicability of IMP analysis in differentiating mixed IPMN from BD-IPMN, but must be validated further in larger studies. IMP profile comparisons with other clinically relevant pancreatic cystic lesions, including mucinous cystic neoplasms and serous cystadenomas, is needed and will expand the diagnostic utility of this technique. A potential limitation is that the peak concentration of certain IMPs in pancreatic cyst fluid may depend on the degree of dysplasia in the cyst. Assessing IMP levels in pancreatic cysts with different grades of dysplasia merits further study. In addition, one of our patients with mixed IPMN had a history of acute pancreatitis, which can have an effect on the IMP levels in the pancreatic fluid. The larger size of pancreatic cysts in the mixed IPMN may have affected IMP levels as well, and this needs further study.
In conclusion, we have successfully identified differentially expressed IMPs in pancreatic cyst fluid of BD-IPMN compared with mixed IPMN using endoscopic collection methods in tandem with cytokine microarray technology. With further validation, our findings may enable the accurate differentiation of mixed IPMN from BD-IPMN using a diagnostic cytokine panel. The advantages of directly investigating pancreatic cyst fluid with this microarray technology include high specificity, small sample volume requirement, cost effectiveness, and complementarity to other detection methods, such as mass spectrometry, ELISA, and western blotting [
Branch duct intraductal papillary mucinous neoplasm
Main duct intraductal papillary mucinous neoplasm
Endoscopic ultrasound-guided fine needle aspiration
Endoscopic retrograde cholangiopancreatography
Inflammatory mediator protein
Computed tomography
Magnetic resonance cholangiopancreatography.
The authors declare no competing interests.
D. L. Conwell and J. A. Paulo equally contributed to this work.
Funds were provided by the American College of Gastroenterology Clinical Research Award (LL, 2011), NIH NIDDK NRSA Fellowship (JP, NIH NIDDK 1 F32 DK085835-01A1), Harvard Digestive Diseases Center (DC, NIH 5 P30 DK034854-24), and NIH NIDDK (DC, 1R21 DK081703-01A2). J. A. Paulo (NIH NIDDK 1 F32 DK085835-01A1).