Title: Gene Expression Profiling Identifies Genes Associated with Invasive Intraductal Papillary Mucinous Neoplasms of the Pancreas
Abstract: 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. 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.1Ohhashi K Murakami F Maruyama M Four cases of mucous secreting pancreatic cancer.Prog Dig Endosc. 1982; 203: 348-351Google Scholar 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.2Loftus Jr, EV Olivares-Pakzad BA Batts KP Adkins MC Stephens DH Sarr MG DiMagno EP Intraductal papillary-mucinous tumors of the pancreas: clinicopathologic features, outcome, and nomenclature. 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Armed Forces Institute of Pathology, Washington, DC1997: 31-144Google Scholar 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.3Sohn TA Yeo CJ Cameron JL Iacobuzio-Donahue CA Hruban RH Lillemoe KD Intraductal papillary mucinous neoplasms of the pancreas: an increasingly recognized clinicopathologic entity.Ann Surg. 2001; 234: 313-321Crossref PubMed Scopus (302) Google Scholar, 6Solcia E Capella C Kloppel G Tumors of the pancreas.in: Rosai J Sobin LH Atlas of Tumor Pathology, series 3. 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Many investigators have applied microarrays to analyze gene expression profiles in invasive pancreatic adenocarcinoma.26Crnogorac-Jurcevic T Efthimiou E Capelli P Blaveri E Baron A Terris B Jones M Tyson K Bassi C Scarpa A Lemoine NR Gene expression profiles of pancreatic cancer and stromal desmoplasia.Oncogene. 2001; 20: 7437-7446Crossref PubMed Scopus (171) Google Scholar, 27Iacobuzio-Donahue CA Maitra A Shen-Ong GL van Heek T Ashfaq R Meyer R Walter K Berg K Hollingsworth MA Cameron JL Yeo CJ Kern SE Goggins M Hruban RH Discovery of novel tumor markers of pancreatic cancer using global gene expression technology.Am J Pathol. 2002; 160: 1239-1249Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, 28Han H Bearss DJ Browne LW Calaluce R Nagle RB Von Hoff DD Identification of differentially expressed genes in pancreatic cancer cells using cDNA microarray.Cancer Res. 2002; 62: 2890-2896PubMed Google Scholar, 29Iacobuzio-Donahue CA Maitra A Olsen M Lowe AW Van Heek NT Rosty C Walter K Sato N Parker A Ashfaq R Jaffee E Ryu B Jones J Eshleman JR Yeo CJ Cameron JL Kern SE Hruban RH Brown PO Goggins M Exploration of global gene expression patterns in pancreatic adenocarcinoma using cDNA microarrays.Am J Pathol. 2003; 162: 1151-1162Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar By contrast, only one report has described gene expression patterns in IPMNs investigated using cDNA microarrays.30Terris B Blaveri E Crnogorac-Jurcevic T Jones M Missiaglia E Ruszniewski P Sauvanet A Lemoine NR Characterization of gene expression profiles in intraductal papillary-mucinous tumors of the pancreas.Am J Pathol. 2002; 160: 1745-1754Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar 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 MethodsTissue Samples and CellsTwelve 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.6Solcia E Capella C Kloppel G Tumors of the pancreas.in: Rosai J Sobin LH Atlas of Tumor Pathology, series 3. Armed Forces Institute of Pathology, Washington, DC1997: 31-144Google Scholar 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 HybridizationAll 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 HybridizationFirst- 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 DataHierarchical 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,31Wodicka L Dong H Mittmann M Ho MH Lockhart DJ Genome-wide expression monitoring in Saccharomyces cerevisiae.Nature Biotechnol. 1997; 15: 1359-1367Crossref PubMed Scopus (856) Google Scholar 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.ImmunohistochemistryImmunohistochemical 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 AnalysisStatistical analysis was performed using Fisher's exact probability test or Mann-Whitney U nonparametric test. Differences were considered significant at P < 0.05.ResultsClinicopathological Characteristics of IPMN PatientsThe 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 IPMNsUsing 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.Figure 2Hierarchical cluster analysis of 12 IPMNs with or without an associated invasive carcinoma and 2 normal pancreatic ductal epithelial samples.View Large Image Figure ViewerDownload Hi-res image Download (PPT)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).32Yonezawa S Horinouchi M Osako M Kubo M Takao S Arimura Y Nagata K Tanaka S Sakoda K Aikou T Sato E Gene expression of gastric type mucin (MUC5AC) in pancreatic tumors: its relationship with the biological behavior of the tumor.Pathol Int. 1999; 49: 45-54Crossref PubMed Scopus (113) Google Scholar, 33Luttges J Zamboni G Longnecker D Kloppel G The immunohistochemical mucin expression pattern distinguishes different types of intraductal papillary mucinous neoplasms of the pancreas and determines their relationship to mucinous noncystic carcinoma and ductal adenocarcinoma.Am J Surg Pathol. 2001; 25: 942-948Crossref PubMed Scopus (226) Google Scholar, 34Terris B Dubois S Buisine MP Sauvanet A Ruszniewski P Aubert JP Porchet N Couvelard A Degott C Flejou JF Mucin gene expression in intraductal papillary-mucinous pancreatic tumours and related lesions.J Pathol. 2002; 197: 632-637Crossref PubMed Scopus (101) Google Scholar, 35Adsay NV Merati K Andea A Sarkar F Hruban RH Wilentz RE Goggins M Iocobuzio-Donahue C Longnecker DS Klimstra DS The dichotomy in the preinvasive neoplasia to invasive carcinoma sequence in the pancreas: differential expression of MUC1 and MUC2 supports the existence of two separate pathways o