Low-grade Intraductal Papillary Mucinous Neoplasms Research Articles

Abstract 3121: Characterization of immune profiling of pancreatic intraductal papillary mucinous neoplasm using multiplex immunofluorescence and image analysis approaches

Abstract Pancreatic intraductal papillary mucinous neoplasms (IPMN) are cystic mucin-secreting tumors that arise within the pancreatic ductal system and are considered precursor lesions of pancreatic ductal adenocarcinoma. Profiling the immune infiltrate of IPMNs is essential to understand the immune mechanisms associated with the progression of IPMN. The aim of this study was to characterize the immune profiling of IPMN. We examined 31 formalin-fixed and paraffin-embedded tumor samples from surgically resected IPMN. The diagnostic slides were histologically reviewed by 2 expert pathologists in pancreas and classified as low-grade IPMN (low to intermediate grade dysplasia, N=11), high-grade IPMN (high grade dysplasia, N=18) and IPMN with invasive adenocarcinoma (N=2). Multiplex immunofluorescence (mIF) was used to characterize the immune profiling using two panels: Panel 1: PD-L1, PD1, CD3 (pan T cells), CD8 (T-cytotoxic), CD68 (macrophages), CK (AE1/AE3), DAPI. Panel 2: CD20 (B cells), CD45RO (T-memory), CD57 (NK), Granzyme B (NK and cytotoxic), FOXP3 (T reg), CK (AE1/AE3), DAPI. The slides were scanned using the Vectra multispectral microscope and analyzed by the InForm software (PerkinElmer). We quantified and co-localized immune phenotypes (epithelial cells PD-L1+; Total T-cell lymphocytes; T-cells antigen-experienced; Cytotoxic T-cells; Total macrophages; Macrophages PD-L1+; Total B-cell lymphocytes; Memory T cells; Granzyme B+ cells; Regulatory T cells; NK cells) in low grade dysplasia (LGD), high grade dysplasia (HGD), and invasive carcinoma areas in epithelial and stromal compartments. The Wilcoxon rank sum test or the Wilcoxon singed-rank test was used to test the equality of medians between two independent groups or paired samples, respectively. In all patients, HGD areas showed significantly higher density of macrophages (P= 0.041) and lower density of B-cell lymphocytes (P= 0.028) compared with LGD areas in the epithelial/stromal and epithelial compartments, respectively. Similar differences were observed when patients with high-grade IPMN (n=18) were analyzed for immune infiltrates in the HGD areas compared with LGD areas in the same tumor samples: HGD areas showed significantly higher density of macrophages (P= 0.035) and lower density of B-cell lymphocytes (P= 0.031). Patients with high-grade IPMN had significantly lower densities of B-cell lymphocytes (P= 0.011), T-cell lymphocytes (P= 0.034), and memory T-cells (P= 0.031) in the HGD areas when compared with LGD areas from patients with low-grade IPMN, in the epithelial compartment. No significant differences were found between LGD areas from patients who had high-grade IPMN and LGD areas from patients with low-grade IPMN. In conclusion, we characterized the immune landscape of IPMN and identified immune cell biomarkers associated with progression of IPMN. Citation Format: Luisa M. Solis, Naohiro Uraoka, Edwin Roger Parra, Yu Shen, Wei Wei, Mei Jiang, Barbara Mino, Kajsa Affolter, Courtney L. Scaife, Michele T. Yip-Schneider, C. Max Schmidt, Matthew Firpo, Sean Mulvihill, Eugene J. Koay,, Huamin Wang, Ignacio I. Wistuba, Anirban Maitra, Subrata Sen. Characterization of immune profiling of pancreatic intraductal papillary mucinous neoplasm using multiplex immunofluorescence and image analysis approaches [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3121.

A Functional Spatial Analysis Platform for Discovery of Immunological Interactions Predictive of Low-Grade to High-Grade Transition of Pancreatic Intraductal Papillary Mucinous Neoplasms.

Intraductal papillary mucinous neoplasms (IPMNs), critical precursors of the devastating tumor pancreatic ductal adenocarcinoma (PDAC), are poorly understood in the pancreatic cancer community. Researchers have shown that IPMN patients with high-grade dysplasia have a greater risk of subsequent development of PDAC in the remnant pancreas than do patients with low-grade dysplasia. In this study, we built a computational prediction model that encapsulates the spatial cellular interactions in IPMNs that play key roles in the transformation of low-grade IPMN cysts to high-grade cysts en route to PDAC. Using multiplex immunofluorescent images of IPMN cysts, we adopted algorithms from spatial statistics and functional data analysis to create metrics that summarize the spatial interactions in IPMNs. We showed that an ensemble of models learned using these spatial metrics can robustly predict, with high accuracy, (1) the dysplasia grade (low vs high grade) and (2) the risk of a low-grade cyst progressing to a high-grade cyst. We obtained high classification accuracies on both tasks, with areas under the curve of 0.81 (95% confidence interval: 0.71-0.9) for task 1 and 0.81 (95% confidence interval: 0.7-0.94) for task 2. To the best of our knowledge, this is the first application of an ensemble machine learning approach for discovering critical cellular spatial interactions in IPMNs using imaging data. We envision that our work can be used as a risk assessment tool for patients diagnosed with IPMNs and facilitate greater understanding and investigation of the cellular interactions that cause transition of IPMNs to PDAC.

DNA Cytometry for Differentiation Between Low- and Medium-grade Dysplasia in Intraductal Papillary Mucinous Neoplasms.

The indication for resection of cystic pancreatic lesions is usually performed by sectional imaging criteria, such as the Sendai criteria. The aim of this study was to analyze a possible correlation between DNA cytometry and Sendai criteria for the differentiation between low-grade intraductal papillary mucinous neoplasms (IPMN-A) and medium-grade dysplasia (IPMN-B). Histopathological analysis, DNA index and preoperative Sendai criteria were determined in 16 patients who underwent pancreatic resection for IPMN. All patients with IPMN-B showed aneuploid histograms with DNA indices ≥1.3, whereas three out of four patients with IPMN-A had diploid DNA indices ≤1.3. All 11 patients with one or more high-risk stigmata and aneuploid histograms had IPMN-Bs, whereas both patients who were Sendai-negative and diploid in the DNA analysis had an IPMN-A. DNA index may be an important diagnostic tool for the differentiation of different IPMN types beyond the traditional Sendai criteria.

Expression of GRP78 protein in pancreatic ductal adenocarcinoma and intraductal papillary mucinous neoplasm.

323 Background: Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis due to late detection, aggressive biology and resistance to chemotherapy. Markers for early detection of PDAC and mediators of chemoresistance are poorly understood. GRP78 is a major endoplasmic reticulum (ER) chaperone protein critical for protein quality control of the ER. Increased GRP78 expression promotes in vitro cancer cell survival, progression, and chemoresistance. This study aims to examine expression of GRP78 protein in PDAC and intraductal papillary mucinous neoplasm (IPMN) compared to normal pancreatic ducts using immunohistochemistry (IHC). Methods: The expression of GRP78 was assessed using IHC in 93 sections of formalin-fixed paraffin-embedded tissue: normal ducts (38), low grade IPMN (IPMN-LG, 26), high grade IPMN (IPMN-HG, 9), and PDAC (20). Immunostaining intensity of GRP78 protein was categorized as no or weak staining (0-1+) and strong staining (2-3+). Fisher’s exact test with two tails was performed using the GraphPad statistical software. Results: GRP78 expression was identified in the cytoplasm of normal pancreatic ducts, IPMN and PDAC with a fine granular pattern. All PDAC and IPMN-HG showed strong expression of GRP78 while the normal ductal cells showed only minimal expression of GRP78. 73% of IPMN-LG expresses GRP78 strongly. Statistical analysis revealed a significant difference in GRP78 expression level between normal ductal cells and all three pathological conditions including PDCA, IPMN-HG and IPMN-LG (all p < 0.0001). Conclusions: This study shows that the expression level of GRP78 is significantly increased in PDAC, IPMN-HG and IPMN-LG compared to normal ductal cells. There appears to be a progressive increase in GRP78 expression from IPMN-LG to IPMN-HG and invasive PDAC. Increased GRP78 expression may be a marker for those at risk of developing PDAC. Changes in GRP78 expression over time and its role in PDAC chemoresistance should be further assessed. [Table: see text]

Distinct patterns of peritumoral histological findings in subtypes of intraductal papillary mucinous neoplasms of the pancreas.

Intraductal papillary mucinous neoplasms (IPMNs) of the pancreas are primary pancreatic neoplasms that can act as precursors to invasive adenocarcinoma of the pancreas. The peritumoral stroma has been increasingly recognized to play an important role in many types of tumors. Therefore, to investigate the clinicopathological significance of the peritumoral stroma in IPMNs, we examined the histological features of the peritumoral stroma in each subtype and histological grade of IPMNs. Eighty-two patients with IPMN, who underwent surgical resection, were reviewed clinicopathologically. Intraductal papillary mucinous neoplasms (86 lesions in total) were histologically subclassified into gastric (n = 51), intestinal (n = 22), pancreatobiliary (n = 11), and oncocytic (n = 2) subtypes. Peritumoral histological features between the gastric and intestinal subtypes were evaluated by each histological grade. The results showed that subepithelial edema and inflammatory cell infiltration were more commonly observed in the gastric subtype (74% and 79%, respectively) than in the intestinal subtype (12% and 25%, respectively) of low-grade IPMNs. On the other hand, mucus lakes were more commonly observed in the intestinal subtype (100%) than in the gastric subtype (0%) of high-grade IPMNs. In addition, pancreatobiliary subtype IPMNs tended to exhibit acute inflammation such as neutrophil predominance. This study showed that peritumoral histological features differed among subtypes of IPMNs from low-grade tumors. These differences suggest the possibility that each subtype of IPMNs has a distinct mechanism from an early stage of tumor progression, which is reflected in the properties of the peritumoral stroma.

Implications of imaging criteria for the management and treatment of intraductal papillary mucinous neoplasms - benign versus malignant findings.

Evaluation of computed tomography (CT) and magnetic resonance imaging (MRI) for differentiation of pancreatic intraductal papillary mucinous neoplasm (IPMN) subtypes based on objective imaging criteria. Fifty-eight patients with 60 histologically confirmed IPMNs were included in this retrospective study. Eighty-three imaging studies (CT,n = 42; MRI,n = 41) were analysed by three independent blinded observers (O1-O3), using established imaging criteria to assess likelihood of malignancy (-5, very likely benign; 5, very likely malignant) and histological subtype (i.e., low-grade (LGD), moderate-grade (MGD), high-grade dysplasia (HGD), early invasive carcinoma (IPMC), solid carcinoma (CA) arising from IPMN). Forty-one benign (LGD IPMN,n = 20; MGD IPMN,n = 21) and 19 malignant (HGD IPMN,n = 3; IPMC,n = 6; solid CA,n = 10) IPMNs located in the main duct (n = 6), branch duct (n = 37), or both (n = 17) were evaluated. Overall accuracy of differentiation between benign and malignant IPMNs was 86/92 % (CT/MRI). Exclusion of overtly malignant cases (solid CA) resulted in overall accuracy of 83/90 % (CT/MRI). The presence of mural nodules and ductal lesion size ≥30 mm were significant indicators of malignancy (p = 0.02 and p < 0.001, respectively). Invasive IPMN can be identified with high confidence and sensitivity using CT and MRI. The diagnostic problem that remains is the accurate radiological differentiation of premalignant and non-invasive subtypes. • CT and MRI can differentiate benign from malignant forms of IPMN. • Identifying (pre)malignant histological IPMN subtypes by CT and MRI is difficult. • Overall, diagnostic performance with MRI was slightly (not significantly) superior to CT.

Epithelial-to-mesenchymal transition (EMT) in intraductal papillary mucinous neoplasm (IPMN) is associated with high tumor grade and adverse outcomes.

Epithelial-to-mesenchymal transition (EMT) is generally associated with increased tumor aggressiveness and poor prognosis. We evaluated EMT characteristics in intraductal papillary mucinous neoplasm (IPMN) tumor specimens and their potential role as biomarkers for malignancy, metastasis, and adverse patient outcomes. IPMN surgical specimens were identified and reviewed by two gastrointestinal pathologists. Immunohistochemical analysis of E-cadherin, vimentin, and ZEB-1 was performed. Samples were linked to clinicopathologic and outcome data for these patients. Western blot test was used to evaluate ZEB-1 expression in IPMN samples; 846 human miRNAs were profiled, and EMT-related differentially expressed miRNAs were validated using quantitative real-time polymerase chain reaction. Fifty-eight IPMN specimens and five normal pancreatic tissue samples were immunohistochemically stained and scored. E-cadherin expression was significantly lower in malignant versus low-grade IPMN (p < 0.05). Vimentin expression was increased in malignant IPMN tumor samples (p < 0.05). EMT was associated with increased lymph node metastasis and decreased survival of malignant IPMN patients (p < 0.05). ZEB-1, an imperative EMT regulator, was exclusively expressed by malignant IPMN tumors. miRNA hierarchical clustering demonstrated grouping of two main IPMN subgroups: low-grade IPMN versus high-grade IPMN and carcinoma. Twenty-four miRNAs were differentially expressed (14 up-regulated, 10 down-regulated). The EMT-regulatory miRNAs, miR-200c and miR-141, were down-regulated (twofold and 1.8-fold decrease, respectively) in malignant versus low-grade IPMN (p < 0.05). EMT may play a role in IPMN tumorigenesis and metastasis. EMT molecular deregulations could be utilized as potential novel biomarkers for the identification of high-risk IPMN patients.

Intranuclear cytoplasmic inclusions are a specific feature of intraductal papillary mucinous neoplasms that distinguish contaminating gastric epithelium

Low-grade intraductal papillary mucinous neoplasms (IPMN) are challenging to diagnose because of an absence of reliable morphologic or immunohistochemical features to distinguish them from contaminating gastric foveolar epithelium. After noting intranuclear cytoplasmic inclusions (ICIs) in some cases of IPMN, we investigated whether ICIs could be used as a specific feature to distinguish IPMN from gastric foveolar epithelium. A consecutive cohort of 61 transduodenal endoscopic fine-needle aspirations of histologically or clinically verified pancreatic IPMNs without high-grade dysplasia from 2005 to 2012 were identified. A control cohort of 24 endoscopic fine-needle aspirations containing gastric epithelium was selected from transgastric specimens of nonpancreatic targets from the same period. Every fragment of mucinous epithelium in the 2 cohorts was examined in alcohol-fixed and cell block sections at high magnification to identify ICIs. ICIs were observed in 31% (19 of 61) of cases in mucinous epithelial fragments obtained by fine-needle aspirations from low-grade IPMNs. When present, they were seen in about 1% of all cells. No ICIs were identified in the control cohort of 24 patients with normal gastric epithelium (P = 0.001 Fisher exact test). BRAF mutation (V600E) testing was performed on 5 IPMN cases, and was negative in all cases including 2 with and 3 without ICIs. KRAS mutation testing was performed on 9 cases of IPMN cases. Two cases with ICIs tested positive for KRAS mutations. Four cases without ICIs also tested positive, and 3 cases without ICIs tested negative. ICIs are a specific morphologic feature found in about one third of low-grade IPMNs, but absent in gastric foveolar epithelium. There is no obvious molecular correlate with the presence of ICIs.

Loss of PTEN Expression Is Associated with Poor Prognosis in Patients with Intraductal Papillary Mucinous Neoplasms of the Pancreas

Previously, we reported PIK3CA gene mutations in high-grade intraductal papillary mucinous neoplasms (IPMN). However, the contribution of phosphatidylinositol-3 kinase pathway (PI3K) dysregulation to pancreatic carcinogenesis is not fully understood and its prognostic value unknown. We investigated the dysregulation of the PI3K signaling pathway in IPMN and its clinical implication. Thirty-six IPMN specimens were examined by novel mutant-enriched sequencing methods for hot-spot mutations in the PIK3CA and AKT1 genes. PIK3CA and AKT1 gene amplifications and loss of heterozygosity at the PTEN locus were also evaluated. In addition, the expression levels of PDPK1/PDK1, PTEN, and Ki67 were analyzed by immunohistochemistry. Three cases carrying the E17K mutation in the AKT1 gene and one case harboring the H1047R mutation in the PIK3CA gene were detected among the 36 cases. PDK1 was significantly overexpressed in the high-grade IPMN versus low-grade IPMN (P = 0.034) and in pancreatic and intestinal-type of IPMN versus gastric-type of IPMN (P = 0.020). Loss of PTEN expression was strongly associated with presence of invasive carcinoma and poor survival in these IPMN patients (P = 0.014). This is the first report of AKT1 mutations in IPMN. Our data indicate that oncogenic activation of the PI3K pathway can contribute to the progression of IPMN, in particular loss of PTEN expression. This finding suggests the potential employment of PI3K pathway-targeted therapies for IPMN patients. The incorporation of PTEN expression status in making surgical decisions may also benefit IPMN patients and should warrant further investigation.

Abstract LB-70: Tackling a clinical challenge: Using microRNAs to differentiate between low-and high-risk Pancreatic Cysts .

Abstract Background: Intraductal papillary mucinous neoplasms (IPMNs) are cystic pancreatic cancer (PC) precursors that are increasingly being detected incidentally by cross-sectional imaging. IPMNs harbor potential for invasive malignancy depending on the degree of histologic dysplasia which ranges from low to high grade, yet the only way to determine disease severity is through surgical resection and histopathologic investigation. Mi(cro)RNAs, non-coding RNAs that regulate one-third of protein coding genes, are attractive candidate biomarkers of early pancreatic malignancy because they are detectable in tissue and blood in a stable form, making them amenable to reliable measurement, and they have been implicated in the development and progression of PC. The goal of our pilot project was to discover and characterize a miRNA signature that accurately differentiates between low-grade IPMNs that merit continued surveillance and high-grade IPMNs that warrant immediate surgical resection. Methods: In our discovery phase, we microdissected formalin-fixed paraffin-embedded tissue (FFPE) and isolated total RNA from 28 pathologically-confirmed IPMNs (9 low-grade and 19 high-grade) surgically resected at our institution between 1999 and 2011, and evaluated the expression of 378 mature miRNAs using high-throughput Taqman Low Density Arrays. After normalization using the endogenous control RNU44, a rank-sum test was performed to compare the expression between the two groups for each miRNA. Results: Using a false discovery rate of 10%, there were 13 differentially expressed miRNAs with statistical significance. The top candidates include miR-100 (P=1.6 x10−3), miR-99b and miR-99a (P=2.7 x10−3), miR-342-3p and miR-126 (P = 3.7 x10−3), and miR-130a (P = 3.7 x10−3). Several of these miRNAs were highlighted in a recent investigation of FFPE tissue and cyst fluid from patients with IPMNs and other pancreatic cysts, demonstrating consistency of findings. Furthermore, the expression level of the top candidate miRNAs was down-regulated in high-grade compared to low-grade IPMN tissue, in line with recent data supporting a role for these miRNAs as tumor suppressors and regulators of key genes that contribute to cell proliferation and invasion in pancreatic and other malignancies. Analysis is underway to evaluate the expression of the top candidate miRNAs in an independent set of high- and low-grade IPMN tissue specimens, and to correlate miRNA expression with other possible clinical predictors of malignant potential. Conclusions: Although preliminary, our findings suggest that miRNAs may serve as a diagnostic adjunct for stratifying patients with IPMNs for continued surveillance or surgical resection. Citation Format: Jennifer Permuth Wey, Susan McCarthy, Y. Ann Chen, Kate Fisher, Agnieszka Kasprzak, Mark Lloyd, Xiaotao Qu, Timothy Yeatman, Jason Klapman, Domenico Coppola, Mokenge Malafa. Tackling a clinical challenge: Using microRNAs to differentiate between low-and high-risk Pancreatic Cysts . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-70. doi:10.1158/1538-7445.AM2013-LB-70

Mutant TP53 in Duodenal Samples of Pancreatic Juice From Patients With Pancreatic Cancer or High-Grade Dysplasia

Imaging tests can identify patients with pancreatic neoplastic cysts but not microscopic dysplasia. We investigated whether mutant TP53 can be detected in duodenal samples of secretin-stimulated pancreatic juice, and whether this assay can be used to screen for high-grade dysplasia and invasive pancreatic cancer. We determined the prevalence of mutant TP53 in microdissected pancreatic intraepithelial neoplasias (PanINs), intraductal papillary mucinous neoplasms (IPMNs), and invasive adenocarcinomas. TP53 mutations were quantified by digital high-resolution melt-curve analysis and sequencing of secretin-stimulated pancreatic juice samples, collected from duodena of 180 subjects enrolled in Cancer of the Pancreas Screening trials; patients were enrolled because of familial and/or inherited predisposition to pancreatic cancer, or as controls. TP53 mutations were identified in 9.1% of intermediate-grade IPMNs (2 of 22), 17.8% of PanIN-2 (8 of 45), 38.1% of high-grade IPMNs (8 of 21), 47.6% of PanIN-3 (10 of 21), and 75% of invasive pancreatic adenocarcinomas (15 of 20); no TP53 mutations were found in PanIN-1 lesions or low-grade IPMNs. TP53 mutations were detected in duodenal samples of pancreatic juice from 29 of 43 patients with pancreatic ductal adenocarcinoma (67.4% sensitivity; 95% confidence interval, 0.52-0.80) and 4 of 8 patients with high-grade lesions (PanIN-3 and high-grade IPMN). No TP53 mutations were identified in samples from 58 controls or 55 screened individuals without evidence of advanced lesions. We detected mutant TP53 in secretin-stimulated pancreatic juice samples collected from duodena of patients with high-grade dysplasia or invasive pancreatic cancer. Tests for mutant TP53 might be developed to improve the diagnosis of and screening for pancreatic cancer and high-grade dysplasia. Clinical Trial numbers: NCT00438906 and NCT00714701.

MiRNA Biomarkers in Cyst Fluid Augment the Diagnosis and Management of Pancreatic Cysts

The diagnosis of pancreatic cystic lesions has increased dramatically. Most are benign, whereas some, such as intraductal papillary mucinous neoplasms (IPMN), represent precursors of pancreatic adenocarcinoma. Therapeutic stratification of IPMNs is challenging without precise information on dysplasia grade and presence of invasion. We assessed the diagnostic benefit of using miRNAs as biomarkers in pancreatic cyst fluid, focusing on IPMNs because of their frequency and malignant potential. RNA was extracted from 55 microdissected formalin-fixed, paraffin-embedded (FFPE) IPMN specimens, and 65 cyst fluid specimens aspirated following surgical resection. Expression of 750 miRNAs was evaluated with TaqMan miRNA Arrays using 22 FFPE and 15 cyst fluid specimens. Differential expression of selected miRNA candidates was validated in 33 FFPE and 50 cyst fluid specimens using TaqMan miRNA Assays. We identified 26 and 37 candidate miRNAs that distinguish low-grade from high-grade IPMNs using FFPE and cyst fluid specimens, respectively. A subset of 18 miRNAs, selected from FFPE and cyst fluid data, separated high-grade IPMNs from low-grade IPMNs, serous cystadenomas (SCA) and uncommon cysts, such as solid pseudopapillary neoplasms (SPN) and cystic pancreatic neuroendocrine tumors (PanNET). A logistic regression model using nine miRNAs allowed prediction of cyst pathology implying resection (high-grade IPMNs, PanNETs, and SPNs) versus conservative management (low-grade IPMNs, SCAs), with a sensitivity of 89%, a specificity of 100%, and area under the curve of 1. We found candidate miRNAs that helped identify patients with high-grade IPMN and exclude nonmucinous cysts. These classifiers will require validation in a prospective setting to ultimately confirm their clinical usefulness.

Gene Expression Changes Associated With the Progression of Intraductal Papillary Mucinous Neoplasms

The diagnosis of high-grade intraductal papillary mucinous neoplasm (IPMN) is difficult to distinguish from low-grade IPMN. The aim of this study was to identify potential markers for the discrimination of high-grade and invasive (HgInv) IPMN from low- and moderate-grade dysplasia IPMN. Laser capture microdissection was used to isolate distinct foci of low-grade, moderate-grade, high-grade, and invasive IPMN from paraffin-embedded archival tissue from 14 patients who underwent resection for IPMN. Most samples included multiple grades in the same specimen. Affymetrix Human Exon microarrays were used to compare low- and moderate-grade dysplasia IPMN with HgInv IPMN. Sixty-two genes were identified as showing significant changes in expression (P ≤ 0.05 and a 2-fold cutoff), including up-regulation of 41 in HgInv IPMN. Changes in gene expression are associated with biological processes related to malignant behavior including cell motion, cell proliferation, response to hypoxia, and epithelial-to-mesenchymal transition. In addition, altered signaling in several transforming growth factor β-related pathways was exhibited in the progression of IPMN to malignancy. This study identifies a set of genes associated with the progression of IPMN to malignancy. These genes are potential markers that could be used to identify IPMN requiring surgical resection.

Outcomes of Primary Surveillance for Intraductal Papillary Mucinous Neoplasm

BackgroundLimited data are available regarding the natural history of patients undergoing primary surveillance for intraductal papillary mucinous neoplasm (IPMN). We hypothesize that symptoms, radiologic characteristics, and cytopathology will predict cancer risk during surveillance. MethodsBetween March 2002 and March 2010, 522 patients were diagnosed with IPMN at a single, high-volume institution. Low versus high oncologic risk was stratified prospectively. Patients with under 3 months of surveillance were excluded. ResultsTwo hundred ninety-two patients underwent primary surveillance for IPMN. Two hundred forty-four (84%) were classified as low-risk IPMN. Mean surveillance duration was 35 (4–99) months. Thirty (12%) patients initially stratified as low-risk developed a new indication for pancreatic resection. Only 28 underwent resection, and pathologic tissue analysis revealed 27 (96%) low-grade IPMN and one (4%) high-grade dysplastic IPMN. Overall, two (1%) patients initially determined to be low-risk developed invasive cancer. Forty-eight (16%) patients stratified as high-risk IPMN were initially managed nonoperatively. Of the 13 (27%) high-risk patients that died during follow-up, two (15%) died from pancreatic cancer. ConclusionsProgression to pancreatic cancer during surveillance for low-risk IPMN was rare. Current indications for resection did not forecast malignancy. Poor operative candidates with high-risk IPMN progressed to invasive cancer more commonly, though a substantial portion succumbed to non-IPMN-related death.

Loss of expression of the SWI/SNF chromatin remodeling subunit BRG1/SMARCA4 is frequently observed in intraductal papillary mucinous neoplasms of the pancreas

A better molecular characterization of intraductal papillary mucinous neoplasm (IPMN), the most frequent cystic precursor lesion of pancreatic adenocarcinoma, may have a pivotal role in its early detection and in the development of effective therapeutic strategies. BRG1, a central component of the chromatin remodeling complex SWI/SNF regulating transcription, is inactive in several malignancies. In this study, we evaluate the Brg1 expression in intraductal papillary mucinous neoplasm to better understand its role in the pancreatic carcinogenesis. Tissue microarrays of 66 surgically resected IPMNs were immunolabeled for the Brg1 protein. Expression patterns were then correlated with clinicopathologic parameters. Normal pancreatic epithelium strongly immunolabeled for Brg1. Reduced Brg1 expression was observed in 32 (53.3%) of the 60 evaluable IPMN lesions and occurred more frequently in high-grade IPMNs (13 of 17 showed loss; 76%) compared to intermediate-grade (15 of 29 showed loss; 52%) and low-grade IPMNs (4 of 14 showed loss; 28%) (P = .03). A complete loss of Brg1 expression was observed in 5 (8.3%) of the 60 lesions. Finally, a decrease in Brg1 protein expression was furthermore found in a low-passage noninvasive IPMN cell line by Western blot analysis. We did not observe correlation between Brg1 expression and IPMN subtype or with location of the cyst. We provide first evidence that Brg1 expression is lost in noninvasive cystic precursor lesions of pancreatic adenocarcinoma.

Outcomes of Primary Surveillance for Intraductal Papillary Mucinous Neoplasm

Limited data are available regarding the natural history of patients undergoing primary surveillance for intraductal papillary mucinous neoplasm (IPMN). We hypothesize that symptoms, radiologic characteristics, and cytopathology will predict cancer risk during surveillance. Between March 2002 and March 2010, 522 patients were diagnosed with IPMN at a single, high-volume institution. Low versus high oncologic risk was stratified prospectively. Patients with under 3 months of surveillance were excluded. Two hundred ninety-two patients underwent primary surveillance for IPMN. Two hundred forty-four (84%) were classified as low-risk IPMN. Mean surveillance duration was 35 (4–99) months. Thirty (12%) patients initially stratified as low-risk developed a new indication for pancreatic resection. Only 28 underwent resection, and pathologic tissue analysis revealed 27 (96%) low-grade IPMN and one (4%) high-grade dysplastic IPMN. Overall, two (1%) patients initially determined to be low-risk developed invasive cancer. Forty-eight (16%) patients stratified as high-risk IPMN were initially managed nonoperatively. Of the 13 (27%) high-risk patients that died during follow-up, two (15%) died from pancreatic cancer. Progression to pancreatic cancer during surveillance for low-risk IPMN was rare. Current indications for resection did not forecast malignancy. Poor operative candidates with high-risk IPMN progressed to invasive cancer more commonly, though a substantial portion succumbed to non-IPMN-related death.

Expression of claudin-4 (CLDN4) mRNA in intraductal papillary mucinous neoplasms of the pancreas

Claudin-4, encoding a protein for tight junction formation and function, is highly overexpressed in pancreatic ductal adenocarcinoma and is also associated with invasive adenocarcinomas arising in intraductal papillary mucinous neoplasms of the pancreas. However, the expression pattern of claudin-4 during neoplastic progression of intraductal papillary mucinous neoplasms remains unknown. Using quantitative real-time reverse transcription-PCR, we analyzed claudin-4 mRNA in a panel of 14 pancreatic cancer cell lines and in formalin-fixed paraffin-embedded tissues from 80 patients with intraductal papillary mucinous neoplasms of different histological grades and papillary subtypes. Increased expression of claudin-4 was confirmed in all the pancreatic cancer cell lines tested as compared with normal ductal epithelial cells and fibroblast cultures. The claudin-4 expression was significantly higher in high-grade intraductal papillary mucinous neoplasms (borderline neoplasm and carcinoma) than in low-grade intraductal papillary mucinous neoplasms (adenoma) (P<0.0001). In addition, claudin-4 mRNA levels were significantly higher in intestinal-type intraductal papillary mucinous neoplasms than in non-intestinal-type intraductal papillary mucinous neoplasms based on papillary subclassification (P<0.0001). Our findings suggest that claudin-4 expression is associated with neoplastic progression of intraductal papillary mucinous neoplasms and, especially, with a distinct pathway to intestinal differentiation.

Transforming Growth Factor α Levels in Pancreatic Fluid

To determine if the level of transforming growth factor α (TGF-α) in the pancreatic fluid (PF) can diagnose intraductal papillary mucinous neoplasm (IPMN) versus other cystic lesions of the pancreas in patients. Pancreatic fluid was prospectively obtained from patients during routine endoscopy and/or operation at Indiana University Hospital. Pancreatic fluid TGF-α levels were analyzed by enzyme-linked immunosorbent assay. Intraductal papillary mucinous neoplasm tissue was also analyzed by TGF-α immunohistochemistry. Sixty-nine fluid samples from 58 patients with the following pathologically confirmed pancreatic disorders were analyzed: IPMN (26 patients), serous cystadenoma (6), mucinous cystic neoplasm (9), pseudocysts (5), non-IPMNY associated pancreatic ductal adenocarcinoma (6), and sphincter of Oddi dysfunction (6). There was no significant difference between the mean PF-TGF-α levels in each category or between different dysplastic grades of IPMN. However, of all the diagnoses examined, only IPMN demonstrated PF-TGF-α levels greater than 95 pg/mL. In low-grade IPMN specimens, TGF-α immunohistochemistry correlated with enzyme-linked immunosorbent assay levels. The mean PF-TGF-α levels are not significantly different in IPMN lesions compared with those in other cystic pancreatic lesions, pancreatic ductal adenocarcinoma, or sphincter of Oddi dysfunction. However, PF-TGF-α levels more than 95 pg/mL may be useful in diagnosing IPMN. This assertion requires prospective validation.

Aberrant methylation of CpG islands in intraductal papillary mucinous neoplasms of the pancreas

Background & Aims: The functional abrogation of several tumor suppressor genes, including p16, DPC4, and p53, is a major mechanism underlying pancreatic ductal carcinogenesis. However, mutational inactivation of these genes is relatively uncommon in intraductal papillary mucinous neoplasms (IPMNs) of the pancreas. We hypothesized that an alternative mechanism for gene inactivation (notably, transcriptional silencing by promoter methylation) could be important in the pathogenesis of IPMNs. Methods: Using methylation-specific polymerase chain reaction, we analyzed the methylation status of 7 CpG islands previously identified as aberrantly methylated in pancreatic adenocarcinoma (including preproenkephalin [ppENK], p16, and thrombospondin 1) in 51 IPMNs of different histologic grades. The relationship between methylation status and expression was evaluated using reverse-transcription polymerase chain reaction for ppENK and immunohistochemistry for p16.Results: We found that more than 80% of the IPMNs exhibited hypermethylation of at least one of these CpG islands. Hypermethylation of ppENK and p16 was detected at a significant higher frequency in high-grade (in situ carcinoma) IPMNs than in low-grade (adenoma/borderline) IPMNs (ppENK, 82% vs. 28%, P = 0.0002; p16, 21% vs. 0%, P = 0.04). Furthermore, the average number of methylated loci was significantly higher in high-grade IPMNs than in low-grade IPMNs (2.4 vs. 0.9; P = 0.0008). Aberrant methylation of ppENK and p16 was associated with loss of expression. Conclusions: These results suggest that de novo methylation of multiple CpG islands is one of the critical pathways that contributes to the malignant progression of IPMNs.GASTROENTEROLOGY 2002;123:365-372