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(American Journal of Pathology. 2004;164:903-914.)
© 2004 American Society for Investigative Pathology

Gene Expression Profiling Identifies Genes Associated with Invasive Intraductal Papillary Mucinous Neoplasms of the Pancreas

Norihiro Sato*, Noriyoshi Fukushima*, Anirban Maitra*, Christine A. Iacobuzio-Donahue*, N. Tjarda van Heek*, John L. Cameron{dagger}, Charles J. Yeo{dagger}{ddagger}, Ralph H. Hruban*{ddagger} and Michael Goggins*{ddagger}§

From the Departments of Pathology,* Surgery,{dagger} Oncology,{ddagger} and Medicine,§ the Johns Hopkins Medical Institutions, Baltimore, Maryland


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 Note Added in Proof
 References
 
The molecular pathology of intraductal papillary mucinous neoplasms (IPMNs) of the pancreas has not been well characterized, and there are no reliable markers to predict the presence of an associated invasive carcinoma in IPMNs. Using oligonucleotide microarrays, we performed a large-scale gene expression profiling of 12 IPMNs with or without an associated invasive carcinoma. A subset of genes identified was validated for the gene expression patterns in a large panel of IPMNs by reverse-transcription polymerase chain reaction and/or immunohistochemistry. A total of 673 transcripts were identified as expressed at significantly higher levels (P < 0.05 and at fivefold or greater) in IPMNs relative to normal pancreatic ductal epithelial samples. Of interest, many of the genes identified as overexpressed in IPMNs have also been previously reported to be highly expressed in infiltrating ductal adenocarcinoma of the pancreas. By analyzing genes overexpressed selectively in IPMNs with an associated invasive carcinoma (n = 7), we also identified a panel of genes potentially associated with the invasive phenotype of the neoplasms. Immunohistochemical validation revealed that claudin 4, CXCR4, S100A4, and mesothelin were expressed at significantly high frequency in invasive IPMNs than in noninvasive IPMNs. Notably, the expression of at least two of the four proteins was observed in 73% of 22 invasive IPMNs but in none of 16 noninvasive IPMNs (P < 0.0001). Our findings suggest that preoperative assessment of gene expression profiles may be able to differentiate invasive from noninvasive IPMNs.

Intraductal papillary mucinous neoplasm (IPMN) of the pancreas was originally identified as a distinct pancreatic neoplasm with a characteristic endoscopic finding of extrusion of mucin through the ampulla of Vater.1 Since the recognition of this entity by the World Health Organization in 1996, IPMNs have been identified with increased frequency and the unique clinical, radiological, and pathological features of IPMNs have been defined.2-5 IPMNs are characterized as having grossly identifiable proliferations of mucin-producing neoplastic epithelium within dilated pancreatic ducts and ductules.6 The intraductal components of IPMNs display a broad spectrum of dysplasia ranging from adenoma to borderline to carcinoma in situ, and ~30% of IPMNs are associated with an infiltrating adenocarcinoma.3,6-8

Although the overall prognosis for patients with IPMNs is better than it is for patients with conventional infiltrating ductal adenocarcinoma of the pancreas, a subset of IPMNs recur, and some patients die of their disease even after surgical resection.2,3,8-12 Among several prognostic factors reported for patients with IPMNs, the presence of an associated invasive carcinoma has been considered the best predictor for a poor clinical outcome.9,11,13-15 In a recent retrospective study with a large series of IPMNs, the 5-year survival for patients with invasive carcinoma was 36% compared with 84.5% for patients without invasive carcinoma.8 However, there are no reliable imaging techniques or molecular markers to predict the presence of such an invasive component in IPMNs.

In contrast to the progress made in our understanding of the molecular genetics of conventional pancreatic ductal adenocarcinoma,16 less is known about the molecular events associated with IPMNs. Genetic alterations in IPMNs have been reported to involve activating point mutations in the K-ras oncogene,17,18 overexpression of the HER-2/neu (c-erbB2) gene product,17,19 loss of heterozygosity at several chromosomal loci,20 and inactivating mutations in the p5317 and STK11/LKB1 tumor-suppressor genes.21 Biallelic inactivation of the p16 and Smad4/DPC4 genes is relatively uncommon in IPMNs and is primarily confined to those neoplasms with high-grade dysplasia or invasive carcinoma.22,23 Recently, we and other investigators have demonstrated that aberrant CpG island hypermethylation of tumor-suppressor genes is a frequent event in IPMNs.24,25

The emergence of microarray technology has enabled us to analyze global gene expression patterns in neoplasms by determining the expression of a large number of transcripts simultaneously. Many investigators have applied microarrays to analyze gene expression profiles in invasive pancreatic adenocarcinoma.26-29 By contrast, only one report has described gene expression patterns in IPMNs investigated using cDNA microarrays.30 Characterization of genes differentially expressed in IPMNs may provide significant insights into the molecular basis of this distinct type of neoplasm. Additionally, the discovery of molecular markers that reliably predict the behavior of IPMNs would be of immediate benefit in the clinical setting. We therefore performed a large-scale gene expression profiling in IPMNs using oligonucleotide microarrays (Affymetrix GeneChip) containing more than 13,000 full-length genes.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 Note Added in Proof
 References
 
Tissue Samples and Cells

Twelve fresh-frozen tissues of IPMNs collected from patients undergoing pancreatic resection at the Johns Hopkins Hospital were selected for the present study based on the availability of sufficient quantities of neoplastic cells. For each case, hematoxylin and eosin (H&E)-stained slides were carefully reviewed and the diagnosis of IPMN was confirmed according to recently established criteria.6 Normal pancreatic duct epithelial cells were selectively microdissected from frozen sections of two resected pancreata using a laser-capture microdissection system (PixCell II; Arcturus, Mountain View, CA). A nonneoplastic cell line established from normal human pancreatic ductal epithelium (HPDE) was kindly provided by Dr. Ming-Sound Tsao (University of Toronto, Toronto, Ontario, Canada). Tissue microarrays (TMAs) of IPMNs were constructed from a total of 38 formalin-fixed, paraffin-embedded blocks of IPMNs using a manual tissue puncher/arrayer (Beecher Instruments, Silver Spring, MD). For each case, two to eight cores punched from the representative areas of IPMNs were arrayed on the TMA blocks. This study was performed with approval of the Johns Hopkins Medical Institutions Joint Committee for Clinical Investigation.

RNA Extraction and Preparation for Array Hybridization

All frozen sections of IPMNs were evaluated with H&E staining and trimmed to enrich the population of neoplastic cells. In IPMNs with an associated infiltrating adenocarcinoma, the intraductal component was selectively dissected for analysis so that we were able to obtain a high neoplastic cellularity of ~80 to 90%. Total RNA was isolated from homogenized frozen IPMNs using Trizol reagent (Invitrogen, Carlsbad, CA), and was purified using the RNeasy mini kit (Qiagen, Valencia, CA). Total RNA was extracted from two frozen samples of microdissected normal ductal epithelial cells using the Picopure RNA isolation kit (Arcturus) according to the manufacturer’s instructions, and was subjected to two rounds of linear amplification using the RiboAmp RNA amplification kit (Arcturus).

Oligonucleotide Array Hybridization

First- and second-stranded cDNA was synthesized from 10 µg of total RNA using T7-(dT)24 primer (Genset Corp., South La Jolla, CA) and SuperScript Choice system (Invitrogen). Labeled cRNA was synthesized from the purified cDNA by in vitro transcription reaction using the BioArray HighYield RNA transcript labeling kit (Enzo Diagnostics, Inc., Farmingdale, NY) at 37°C for 6 hours. The cRNA was fragmented at 94°C for 35 minutes in a fragmentation buffer (40 mmol/L Tris-acetate, pH 8.1, 100 mmol/L potassium acetate, 30 mmol/L magnesium acetate). One of the fragmented cRNA samples was run on the Test3 chip (Affymetrix, Santa Clara, CA) to ensure the quality of target samples. The fragmented cRNA samples were then hybridized to the human genome U133A chips (Affymetrix) at 45°C for 16 hours. The washing and staining procedure was performed in the Affymetrix Fluidics Station according to the manufacturer’s instructions. The probes were then scanned using a laser scanner, and signal intensity for each transcript (background-subtracted and adjusted for noise) and detection call (present, absent, or marginal) were determined using Microarray Suite software 5.0 (Affymetrix).

Analysis of Microarray Data

Hierarchical cluster analysis was performed using dChip (DNA-chip analyzer) software (www.dChip.org) after filtering genes with the greatest variation across all samples (SD/mean >2). The analysis of genes differentially expressed between IPMNs and control (normal pancreatic duct epithelial) samples was performed with fold-change analysis and t-test using the Data Mining Tool software (Affymetrix). Expression data for all probe sets was filtered out to identify transcripts expressed at significantly higher levels (P < 0.05 by t-test and at least fivefold greater) in IPMNs compared to control samples. We also compared gene expression profiles between IPMNs with an associated invasive carcinoma (n = 7) and IPMNs without an associated invasive carcinoma (n = 5) and identified transcripts that were significantly (P < 0.05 by t-test and at least threefold greater) overexpressed selectively in IPMNs with an associated invasive carcinoma relative to control samples. Although it has been demonstrated that expression changes more than twofold are significant in Affymetrix microarrays,31 we used a more stringent cutoff to reduce the number of false-positives.

Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)

We performed RT-PCR to confirm the expression patterns of selected genes in 12 IPMNs (all of which were subjected to array analysis) and in the nonneoplastic HPDE cells. Four µg of total RNA was reverse-transcribed using Superscript II (Invitrogen). PCR reaction was performed as follows: 95°C for 5 minutes; then 35 cycles of 95°C for 20 seconds, 60°C for 20 seconds, and 72°C for 20 seconds; and a final extension of 4 minutes at 72°C. Primer sequences are available on request. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was also amplified in the same PCR reaction to ensure the cDNA integrity.

Immunohistochemistry

Immunohistochemical analysis was performed to validate the differential expression of selected genes on archival IPMN tissue sections or TMAs containing 38 IPMNs. Five-µm sections were cut onto coated slides and deparaffinized by routine techniques. Antigen retrieval was performed in 10 mmol/L of sodium citrate buffer (pH 6.0) heated at 95°C in a steamer for 20 minutes. After blocking endogenous peroxidase activity with a 3% aqueous H2O2 solution for 5 minutes, the sections were incubated with primary antibodies for 30 to 60 minutes. The antibodies used and their dilutions were as follows: S100A4 (1:500; DAKO, Carpinteria, CA), prostate stem cell antigen (PSCA) (1:200, clone 1G8; kindly provided from Dr. Robert E. Reiter, Dept. of Urology, Univ. of California), decay accelerating factor for complement (1:50, CD55, H-319; Santa Cruz Biotechnology, Santa Cruz, CA), TIMP1 (1:20, H-150; Santa Cruz), mesothelin (1:20, clone 5B2; Novocastra Laboratories, Newcastle, UK), CXCR4 (1:50, clone 12G5; Zymed Laboratories, South San Francisco, CA), and claudin 4 (1:400, clone 3E2C1; Zymed Laboratories). Labeling was detected with the Envision Plus Detection Kit (DAKO) following the protocol as suggested by the manufacturer, and all sections were counterstained with hematoxylin. Slides or individual cores on TMAs were scored as either positive (positive staining in >10% of neoplastic cells) or negative. To validate TMA, cases were considered positive if at least one tissue core showed positive immunostaining and negative if none of the tissue cores showed positive labeling.

Statistical Analysis

Statistical analysis was performed using Fisher’s exact probability test or Mann-Whitney U nonparametric test. Differences were considered significant at P < 0.05.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 Note Added in Proof
 References
 
Clinicopathological Characteristics of IPMN Patients

The 12 IPMN cases analyzed for gene expression profiling included six men and six women with a mean age of 72 years (range, 60 to 79 years). These neoplasms arose from the head of the pancreas in nine (75%) cases, body of the pancreas in two cases, and diffusely involved the entire gland in one case. The maximum diameter of the neoplasms ranged from 2.5 to 9.0 cm (mean, 5.1 cm). The intraductal components were classified as carcinoma in situ in all IPMNs, and seven (58%) of the IPMNs were associated with an infiltrating ductal (tubular) adenocarcinoma. Lymph node metastases were identified in five (71%) of the seven invasive IPMNs, and metastatic disease to the liver was observed in one invasive IPMN at the time of surgery.

Identification of Genes Overexpressed in IPMNs

Using high-density oligonucleotide microarrays (Affymetrix human genome U133A chips) with 18,462 gene/expressed sequence tag transcripts, we performed a gene expression profiling in 12 IPMNs and 2 microdissected histologically normal pancreatic duct epithelial samples. For IPMN samples, the intraductal component was selectively dissected for microarray analysis and was estimated to achieve an average neoplastic cellularity of ~80 to 90% (Figure 1) . Analysis of the gene expression profiles of 12 IPMNs and the 2 normal-appearing pancreatic duct samples identified 369 transcripts that had the greatest variation in transcription among the 14 samples (SD/mean >2). Hierarchical cluster analysis of these 369 transcripts identified two major clusters: the first containing four IPMNs and two normal ductal epithelial samples, and the second containing eight IPMNs (Figure 2) . The two normal ductal epithelial samples were clustered in the closest branches in the dendrogram. Four of the seven IPMNs with an associated invasive carcinoma were clustered in the first major branch with remarkable similarity, whereas all of the five IPMNs without an invasive carcinoma were clustered together in the second major cluster, suggesting different gene expression patterns between these two groups.



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  		Figure 1. An example of the intraductal component of IPMN frozen sections dissected for microarray analysis showing a high neoplastic cellularity (stained with hematoxylin).

 


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  		Figure 2. Hierarchical cluster analysis of 12 IPMNs with or without an associated invasive carcinoma and 2 normal pancreatic ductal epithelial samples.

 
The Affymetrix data mining tool was then used to identify transcripts expressed at significantly higher levels (P < 0.05 by t-test and at least fivefold greater) in IPMNs compared to normal ductal epithelial samples. Furthermore, we eliminated transcripts whose expression call was absent in more than 6 of 12 IPMNs. Using these stringent criteria, a total of 673 transcripts were identified as significantly overexpressed in IPMNs relative to nonneoplastic pancreatic ductal epithelium [Table 1 , and full list of transcripts is available at our website (http://www.pathology2.jhu.edu/pancreas/IPMN)]. We also identified 72 transcripts that were significantly (P < 0.05 and at least fivefold) underexpressed in IPMNs compared to control samples, including known genes such as P-glycoprotein (mdr1) (available at http://www.pathology2.jhu.edu/pancreas/IPMN). The large panel of overexpressed transcripts included several genes whose expression pattern in IPMNs has been confirmed in previous publications, such as those encoding for mucins (MUC1 and MUC5AC).32-35 On the other hand, the majority of the genes identified here have not been previously implicated in IPMNs. Functional classification of these genes revealed several groups associated with important biological processes, including immune response (immunoglobulin genes, interferon-related genes, anti-hepatitis A IgG, and others), calcium homeostasis (S100P, S100A4, S100A6, S100A11, annexin 14, and CACNL1A1), cytoskeletal organization (profilin 1, tropomyosin 2, and plectin 1), cell adhesion (E-cadherin and protocadherin1), cell motility (dynamin 2 and hyaluronan-mediated motility receptor), cell-cycle checkpoint (stratifin and GADD34), cell proliferation (melanoma inhibitory activity and insulin-like growth factor binding protein 2), cell death (death-associated protein (DAP) and cell death-regulatory protein GRIM19), signal transudation (tetraspanin TM4-C, TRAF5, and signal sequence receptor, delta), and extracellular matrix remodeling [TIMP1 and urokinase-type plasminogen activator receptor (uPAR)]. Notably, some of the most highly expressed genes in IPMNs (MUC5AC, pepsinogen C, claudin-18, and cathepsin E) encode proteins known to be primarily expressed in normal mucosa of the stomach, suggesting gastric-type differentiation during the evolution of IPMNs.


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  		Table 1. Highly Expressed Genes in IPMNs as Compared with Normal Pancreatic Ductal Epithelium*

 
Of interest, many of the genes identified by our approach have also been implicated in infiltrating ductal adenocarcinoma of the pancreas. For example, among the most highly expressed 100 known genes identified in IPMNs, at least 21 genes have been previously reported to be highly expressed in pancreatic ductal adenocarcinoma. These genes included S100P,27,36 pyruvate kinase, muscle (PKM2),36 mesothelin,37,38 SERPINB5,36 stratifin,29,36 S100A4,38,39 ubiquitin carrier protein (E2-EPF),36 tropomyosin 2 (TPM2),36 prostate stem cell antigen (PSCA),40 coated vesicle membrane protein,38 decay accelerating factor for complement (CD55),29,36 CEACAM5,36 S100A11,28 S100A6,36 and cysteine-rich protein 1.38 Importantly, aberrant expression of many of these genes has been also associated with the malignant phenotype in a variety of nonpancreatic neoplasms, suggesting that these genes may play central roles in the development and progression of IPMNs.

Confirmation of Expression Patterns of Selected Genes by RT-PCR and Immunohistochemistry

Thirteen genes were chosen from the list of overexpressed transcripts for further confirmation of their expression patterns. We first performed RT-PCR to examine the mRNA expression of 11 genes in 12 IPMNs (all of them were analyzed by microarray). The 11 genes examined were S100P, melanoma inhibitory activity (MIA), pepsinogen C (PGC), SERPINA1, lumican, S100A4, cysteine-rich protein 1 (CRIP1), PSCA, mesothelin, annexin 14 (ANX14), and lipocalin 2. As a control, we used the nonneoplastic pancreatic ductal epithelial cell line (HPDE), which demonstrated the gene expression patterns similar to those of microdissected normal ductal epithelium by microarray analysis (Figure 3A) . All 11 genes were confirmed by RT-PCR to be strongly expressed at mRNA levels in most of the 12 IPMNs in contrast to weak or absent expression in HPDE (Figure 3B) . Of note, the RT-PCR analyses on these genes paralleled the results from microarrays. For example, RT-PCR demonstrated abundant mRNA expression of MIA in eight IPMNs whose expression of the corresponding gene was called present by microarray analysis. By contrast, no detectable MIA transcript was found in the remaining four IPMNs and all four of these had an absent call for this gene in the gene expression microarrays.



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  		Figure 3. A: Microarray expression levels of 11 genes selected for RT-PCR validation in the nonneoplastic ductal cell line (HPDE), microdissected normal ductal epithelium (Avg-NPD), and IPMNs (Avg-IPMNs). B: RT-PCR analysis of 11 selected genes in HPDE and 12 IPMNs. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) serves as a RNA control.

 
We next verified the protein expression of five selected genes by immunohistochemistry on TMAs representing 38 IPMNs and on five archival tissue sections containing normal pancreatic ducts. The proteins tested included S100A4, decay accelerating factor for complement (CD55), TIMP1, mesothelin, and PSCA. Representative results are shown in Figure 4 . For each of these proteins, labeling of normal ductal epithelium was weak or minimal. By contrast, moderate to strong labeling was identified in the neoplastic epithelium of IPMNs at varying frequencies. There was no stromal labeling for any of these genes. To confirm the accuracy of the TMA immunohistochemistry, we labeled full tissue sections of five IPMNs and confirmed that the cores adequately represented the labeling pattern of each neoplasm. Overall, positive immunolabeling was identified in 22% of all of the IPMNs tested for S100A4, 49% for CD55, 70% for TIMP1, 25% for mesothelin, and 39% for PSCA. When the labeling pattern of the noninvasive component of the IPMNs was compared between IPMNs with an associated with invasive carcinoma (n = 22) and IPMNs not associated with an invasive carcinoma (n = 16), the expression of S100A4 and mesothelin was found to be more frequency detected in IPMNs with an associated invasive carcinoma than in noninvasive IPMNs (S100A4: 38% versus 0%, P = 0.006; mesothelin: 43% versus 0%; P = 0.005, Fisher’s exact probability test). There were no significant differences in the labeling frequencies of other proteins between invasive and noninvasive IPMNs.



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  		Figure 4. Immunohistochemical validation of five overexpressed genes on IPMN tissue microarrays. Shown are representative tissue cores stained positive for S100A4 (A), CD55 (B), TIMP1 (C), PSCA (D), and mesothelin (E).

 
Identification of Genes Associated with the Invasive Phenotype of IPMNs

Immunohistochemical analysis suggests that mesothelin and S100A4 expression appeared to be restricted to IPMNs with an associated invasive carcinoma. To identify additional genes whose expression was primarily restricted to IPMNs with an associated invasive carcinoma, we first compared the expression profiles between seven IPMNs with an associated invasive carcinoma and two control samples. This analysis identified 1755 transcripts significantly (P < 0.05 by t-test and at least threefold greater) overexpressed in IPMNs with an associated carcinoma relative to nonneoplastic pancreatic duct epithelium. From the 1755 transcripts identified, 1408 transcripts that were also expressed at high levels (threefold or greater) in IPMNs without an associated invasive carcinoma were excluded, leaving 347 transcripts that were preferentially overexpressed in IPMNs with an associated invasive carcinoma. We also excluded transcripts whose expression call was absent in four or more of seven IPMNs with an associated invasive carcinoma. Consequently, we identified 187 transcripts that were overexpressed in IPMNs with an associated invasive carcinoma but not in IPMNs without an associated invasive carcinoma (Table 2 , and full list of transcripts is available at http://www.pathology2.jhu.edu/pancreas/IPMN). In support for our approach, this panel of transcripts included several genes previously implicated in tumor invasion, metastasis, and/or angiogenesis such as chemokine receptor 4 (CXCR4, fusin),41 cyclin D2,42 extracellular matrix protein 1 (ECM1),43 alpha2 integrin,44 lamin B1,45 and S100A4.46,47


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  		Table 2. Representative Subset of Genes Overexpressed Selectively in Invasive IPMNs

 
Among the genes identified as overexpressed in IPMNs with an associated invasive carcinoma, the chemokine receptor CXCR4 has recently attracted considerable interest for its potential role in cell motility, adhesion, invasion, and metastasis of various cancer types.48-50 We therefore determined the expression pattern of CXCR4 in an expanded series of 51 IPMNs (27 IPMNs with an associated invasive carcinoma and 24 IPMNs without an associated invasive carcinoma) by immunohistochemical labeling with an anti-CXCR4 monoclonal antibody. Expression of CXCR4 was not identified in normal ductal epithelium, but was localized to neoplastic epithelial cells in 16 (31%) of 51 IPMNs (Figure 5) . CXCR4 expression was detected at higher frequency in IPMNs with an associated invasive carcinoma than in IPMNs without an associated invasive carcinoma (44% versus 17%, P = 0.04, Fisher’s exact probability test).



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  		Figure 5. Immunohistochemical validation of two selected genes (CXCR4 and claudin 4) associated with the invasive phenotype of IPMNs. CXCR4 is not expressed in normal ductal epithelium (A). Strong immunolabeling of CXCR4 is detected in two cases of invasive IPMNs (B and C), but not in an IPMN associated with colloid carcinoma (D). Claudin 4 is not expressed in normal ductal epithelium (E). Diffuse labeling for claudin 4 is shown in the neoplastic epithelium of invasive IPMNs (F and G), in contrast to negative staining in noninvasive IPMN (H). Note the discrete membrane immunoreactivity of claudin 4 in the neoplastic cells demonstrated by a high-power view (G).

 
We then determined the expression pattern of claudin 4, which was identified as overexpressed in IPMNs with an associated invasive carcinoma, in 51 IPMNs by immunohistochemistry. This gene encodes for a transmembrane protein known to function as a high-affinity receptor for Clostridium perfringens enterotoxin (CPE),51 and claudin 4 has been shown to be overexpressed in invasive adenocarcinoma of the pancreas,27,29,38,52 suggesting a role for this gene in the invasive phenotype during pancreatic neoplastic progression. Normal duct epithelium did not label for claudin 4, whereas membranous immunolabeling was detectable in neoplastic epithelium in 34 (67%) of 51 IPMNs (Figure 5) . Remarkably, positive claudin 4 immunostaining was identified in 100% (27 of 27) of IPMNs with an associated invasive carcinoma but only in 29% (7 of 24) of IPMNs without an associated invasive carcinoma (P < 0.0001), supporting our microarray data.

Protein Expression Profiling of IPMNs

Finally, we investigated by immunohistochemical analyses the protein expression of seven genes (S100A4, CD55, TIMP1, mesothelin, PSCA, claudin 4, and CXCR4) overexpressed in IPMNs using a panel of 38 IPMNs arrayed on TMAs. These IPMNs were classified into adenoma, borderline, carcinoma in situ, and IPMNs with an associated invasive carcinoma according to the established criteria (Figure 6) .6 Among the IPMNs without an associated invasive carcinoma, there was no significant difference observed in the protein expression patterns of these genes between adenoma (n = 4), borderline (n = 5), and carcinoma in situ IPMNs (n = 7). We also classified the invasive component of IPMNs with an associated invasive carcinoma into colloid-type carcinoma (n = 6) or tubular-type carcinoma (n = 16) to determine whether different gene expression patterns existed in these subtypes of invasive carcinoma associated with IPMNs. The expression of mesothelin and CXCR4 was more frequently detected in the noninvasive components of IPMNs with an associated tubular carcinoma than in those with an associated colloid carcinoma, although the difference did not reach statistical significance. Overall, the average number of overexpressed genes was significantly higher in IPMNs with an associated invasive carcinoma than in IPMNs without an associated invasive carcinoma (4.0 versus 1.8; Mann-Whitney U nonparametric test, P = 0.0005). Notably, the expression of at least two of the four proteins (S100A4, mesothelin, claudin 4, and CXCR4) was commonly observed in IPMNs with an associated invasive carcinoma but was not seen in IPMNs without an associated invasive carcinoma; 16 (73%) of 22 invasive IPMNs expressed two or more of these four proteins, compared to none of 16 noninvasive IPMNs (P < 0.0001) (Figure 6) . These results suggest that the assessment of gene expression profiles of IPMNs could potentially be used in the preoperative setting to help identify IPMNs with an associated invasive carcinoma.



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  		Figure 6. Examples for histological classification of IPMNs (left, H&E staining) and expression profiles of a panel of 38 IPMNs on TMAs determined by immunohistochemical analyses of all of the seven genes tested (right). Filled box and open box indicate positive and negative immunolabeling, respectively. ND, not determined.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
Note Added in Proof
 References
 
In the present study, we have characterized a global gene expression profile of IPMNs using a high-throughput approach based on Affymetrix oligonucleotide microarrays featuring more than 18,000 unique transcripts. Although a gene expression study of IPMNs has been recently reported using a cDNA array of ~5000 genes,30 the oligonucleotide microarray platform enabled us to identify a large number of previously unreported genes overexpressed in IPMNs. To identify overexpressed genes, we compared gene expression profiles from IPMNs with those of nonneoplastic pancreatic ductal epithelial cells selectively microdissected by laser-capture microdissection and only defined overexpression if there was a mean fivefold difference in expression levels between groups and if this difference was statistically significant. Because the intraductal components of IPMNs dissected for our microarray analysis were highly enriched (~80% to 90%) for neoplastic epithelium, its with microdissected pancreatic ductal cells minimized the detection of "falsely elevated" genes that commonly arise when one compares tissue types with different proportions of epithelial and other cell types. Using RT-PCR and immunohistochemistry, we were able to confirm the expression patterns of all of the 13 selected genes identified as overexpressed by gene expression microarray analysis. Notably, many of the overexpressed genes identified by our analyses participate in important biological processes and have been previously implicated in a variety of other tumor types, suggesting that many of these genes contribute to the neoplastic evolution, malignant transformation, and invasive progression of IPMNs.

IPMNs are usually slow-growing neoplasms and generally less aggressive than conventional ductal adenocarcinoma of the pancreas, leading to the hypothesis that the molecular alterations of IPMNs differ from those of infiltrating ductal adenocarcinoma.3 In support for this view, previous studies have shown that inactivation of several tumor-suppressor genes such as p53 and DPC4/Smad4 occur less frequently in IPMNs than in infiltrating pancreatic ductal adenocarcinoma.17,22 By contrast, a recent immunohistochemical study demonstrated that inactivation of STK11/LKB1 was more common in IPMNs than in pancreatic ductal adenocarcinoma.53 Therefore, it is possible that IPMNs and pancreatic adenocarcinoma may differ in their expression profiles particularly those genes downstream of pancreatic cancer tumor suppressor genes. Currently, however, little is known about the difference in gene expression profiles between IPMNs and infiltrating pancreatic ductal adenocarcinoma because of the lack of information on gene expression patterns in IPMNs. In the present study, we find that many of the genes identified as overexpressed in IPMNs have also been previously reported to be highly expressed in invasive pancreatic ductal adenocarcinoma. This raises the possibility that these genes are common targets involved in the neoplastic progression of pancreatic ductal system and that IPMN and pancreatic adenocarcinoma share some gene expression pathways. Although IPMNs and pancreatic intraepithelial neoplasias (PanINs) share similar histological features and both are recognized as precursors to invasive adenocarcinoma,7,54 accumulating evidence suggests that IPMNs and PanINs represent two distinct types of intraductal neoplasia. For example, IPMNs and PanINs display different staining patterns for DPC4/Smad422,55 and MUC2,33,35,56 suggesting a fundamental difference in the molecular pathways leading to these intraductal neoplasias of the pancreas.

We also found that some of the most highly expressed genes in IPMNs are primarily expressed in normal mucosa of the stomach, including MUC5AC, pepsinogen C, claudin-18, and cathepsin E. The concomitant overexpression of multiple gastric-related genes suggests differentiation into a gastric-type epithelial phenotype during the neoplastic evolution of IPMNs. Importantly, expression of these gastric-type markers has been linked to a better prognosis in pancreatic and other neoplasms. For example, it has been shown that patients with pancreatic neoplasms expressing MUC5AC mRNA have a significantly better prognosis than those with no MUC5AC mRNA expression.32 Furthermore, pepsinogen C is known to be a strong predictor for favorable outcome in patients with various cancers, including breast,57 gastric,58 and pancreatic cancer.59 Based on these findings, it is possible that consistent overexpression of these gastric-type proteins in IPMNs is related to their indolent biological behavior and relatively favorable outcome.

Among the genes identified as overexpressed in IPMNs, genes encoding secreted proteins may represent promising candidates to be exploited for diagnosis and/or follow-up of patients with IPMNs. In support for this hypothesis, we have recently demonstrated that HIP/PAP-I, a secreted protein overexpressed in the acini adjacent to the invasive pancreatic ductal adenocarcinoma, can be detected at high levels in pancreatic juice and serum samples from patients with pancreatic cancer.60 In our present analysis, we found the overexpression of several genes encoding secreted proteins [such as melanoma inhibitory activity (MIA), lumican, and putative secreted protein XAG] in IPMNs. Among them, MIA has been recently shown to be a potential serological marker for metastatic or recurrent melanoma.61,62 Further studies are required to evaluate the diagnostic value of these secreted proteins in IPMNs.

Widespread use of screening ultrasound and computed tomography has led to the identification of increasing numbers of patients with asymptomatic cystic lesions of the pancreas, including IPMNs.63 Accurate preoperative assessment of the grade of malignancy in IPMNs is critical to determine the optimal management for these patients and to avoid unnecessary radical pancreatic resection in those patients with low-grade IPMNs (adenoma) especially for patients with concomitant disease for whom surveillance would be the preferred approach to management. At present, however, neither imaging nor laboratory markers can adequately differentiate between benign and malignant IPMNs or detect an invasive focus preoperatively. In the present study, we attempted to identify genes associated with the invasive phenotype of IPMNs. To eliminate the contamination of the nonneoplastic tissues (especially of abundant desmoplastic stroma observed in the invasive component associated with IPMNs), we analyzed the intraductal components for gene expression profiling. Therefore, it is likely that some genes that are expressed specifically in the invasive components of IPMNs will be missed using this strategy. We hypothesized that the gene expression profile of the intraductal (noninvasive) components of IPMNs may reflect the invasive potential of these neoplasms. This hypothesis is supported by recent reports that metastatic potential of breast and other solid tumors could be reliably predicted from the gene expression profile of the primary tumor.45,64 Indeed, we were able to identify a large panel of genes overexpressed selectively in the intraductal components of IPMNs with an associated invasive carcinoma, and many of these genes are functionally important for tumor invasion and metastasis, including S100A4, CXCR4, and claudin 4. For example, inhibition of S100A4 expression in osteosarcoma cells with a hammerhead ribozyme results in decreased invasive properties in vitro,46 whereas transfection of S100A4 produces metastatic variants of an orthotopic model of bladder cancer.47 Interaction between the chemokine receptor CXCR4 and its ligand CXCL12/SDF-1 has been shown to play a critical role in invasion and metastasis of different types of cancers including breast,48 prostate,49 and small cell lung cancer.50 Of particular interest, claudin 4 has been identified as a high-affinity epithelial receptor for CPE.51 Recently, it has been shown that claudin 4 is overexpressed in pancreatic ductal adenocarcinoma and treatment with CPE results in an acute cytotoxic effect specifically in claudin 4-expressing pancreatic cancer cells.52 Although the therapeutic significance of claudin 4 has not been determined, our findings suggest that claudin 4 and other invasion-associated genes identified could be molecular targets for therapy as well as for diagnosis in patients with IPMNs with an associated invasive carcinoma.

In summary, we have identified a large set of overexpressed genes that shed light on the molecular basis underlying the pathogenesis and progression of IPMNs. Further characterization of these genes/expressed sequence tags may refine our understanding of IPMNs and enhance our ability to diagnose and manage these patients appropriately.


   Note Added in Proof
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 Note Added in Proof
References
 
We have recently added three normal ductal epithelial samples and reanalyzed the gene expression data (available at http://www.pathology2.jhu.edu/pancreas/IPMN).


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  		Table 1A. Continued

 

   Footnotes
 
Address reprint requests to Michael Goggins, M.D., Departments of Pathology, Medicine, and Oncology, the Johns Hopkins Medical Institutions, 632 Ross Bldg., 720 Rutland Ave., Baltimore, MD 21205-2196. E-mail: mgoggins{at}jhmi.edu

Supported by the Specialized Program of Research Excellence (SPORE) in Gastrointestinal Malignancies (grant CA62924), the Michael Rolfe Foundation, and a gift to support pancreatic cancer research from Susan Gurney.

Accepted for publication November 17, 2003.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 Note Added in Proof
 References
 

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