Pancreatic endocrine neoplasms are neoplastic proliferations of islet cells or islet cell precursors and are capable of secreting a variety of synthetic products, including insulin, glucagon, gastrin, and vasoactive intestinal peptide. The biological behavior of pancreatic endocrine neoplasms is often unpredictable, and there are few reliable histopathologic criteria reliably correlating with metastatic ability. We have used the Affymetrix U133 GeneChip set (HG_U133 A and B; Affymetrix; Santa Clara, CA) representing ϳ33,000 characterized transcripts to examine global gene expression profiles from well-differentiated nonmetastatic (n ‫؍‬ 5) and metastatic (n ‫؍‬ 7) pancreatic endocrine neoplasms to determine molecular markers that predict disease progression. Microarray hybridization data were normalized using the GeneLogic GeneExpress Software System to identify differentially up-and down-regulated genes in metastatic versus nonmetastatic pancreatic endocrine neoplasms. Using a 3-fold change in gene expression as a threshold, we have identified 65 overexpressed and 57 underexpressed genes in metastatic pancreatic endocrine neoplasms as compared with nonmetastatic pancreatic endocrine neoplasms. Several classes of genes, including growth factors and growth factorrelated molecules (IGFBP1, IGFBP3, and MET), developmental factors (TBX3 and MEIS2), cytoskeletal factors (␤ 1 tubulin and ACTN2), cholesterol homeostasis mediators (LRP5, SLC27A2, and RXRG), intracellular signaling pathway mediators (DYRK1A, PKIB, and AK2), methyltransferases (MGMT and GAMT), and DNA repair and regulatory molecules (CHEK1 and ZNF198), were identified as differentially over-or underexpressed via this method. Immunohistochemical validation of microarray data were performed for two overexpressed genes, namely, the met protooncogene (MET) and insulin-like growth factor binding protein 3 (IGFBP3) with tissue microarrays of nonmetastatic (n ‫؍‬ 24) and metastatic (n ‫؍‬ 15) pancreatic endocrine neoplasms. Increased expression of IGFBP3 was confirmed in metastatic versus nonmetastatic pancreatic endocrine neoplasms (12 of 15, 80% versus 10 of 24, 42%), as well as in lymph node (6 of 7, 86%) and liver (9 of 9, 100%) metastases. Similarly, overexpression of MET was confirmed in metastatic versus nonmetastatic pancreatic endocrine neoplasms (5 of 15, 33% versus 4 of 24, 17%), as well as in lymph node metastases (4 of 7, 57%) and liver metastases (5 of 9, 56%). The majority of genes that demonstrated altered expression has not been previously identified as differentially expressed in metastatic pancreatic endocrine neoplasm lesions and may therefore represent newly identified molecules in the progression of these lesions.

Performing immunohistochemical analysis on minute lesions is a challenging task, primarily because they frequently disappear when the paraffin block is recutfor immunostaining purposes. This is a particularly common occurrence with prostate biopsy specimens, in which immunostains for high-molecular-weight cytokeratin commonly are used as an adjunct to H&E examination for aiding in the interpretation of minute "suspicious" lesions. We describe an original method designated tissue protection immunohistochemistry, that allows the performance of high-molecular-weight cytokeratin immunostains (or other immunostains) on previously stained H&E slides. The method described does not require destaining of H&E-stained sections, and it allows the preservation of the H&E stain on adjacent levels that may be present on the same slide. The method described requires that the original H&E-stained sections be placed on adhesive slides, but it has the advantages of eliminating the requirement of a paraffin block for immunostaining and eliminating the need for saving intervening unstained sections for possible immunohistochemical analysis.

We extended the results of a previous microarray analysis by immunohistochemical validation of differential protein expression in a series of 57 surgically resected infiltrating ductal pancreatic adenocarcinomas. Two representative genes were examined: sea urchin fascin homolog (overexpressed in both cell lines and primary tumors) and heat shock protein 47 (HSP47; overexpressed in primary tumors only). Protein expression also was evaluated in the precursor lesions of pancreatic cancer pancreatic intraepithelial neoplasia (PanIN), and normal ductal epithelium. Fascin expression was seen in the neoplastic cells of 54 (95%) of 57 ductal adenocarcinomas but not in 49 (94%) of 52 adjacent nonneoplastic epithelium. In the multistep pathogenesis of ductal adenocarcinomas, fascin expression seemed to be a late event, usually present in PanINs 2 and 3. HSP47 expression was almost universal and most intense in the ductal adenocarcinoma-associated stromal desmoplasia (57/57), although 37 cases (65%) also expressed HSP47 in the neoplastic epithelium. HSP47 expression was absent in the majority of nonneoplastic pancreata (46 [88%]). Fascin and HSP47 are novel tumor markers with potential diagnostic and therapeutic implications for pancreatic carcinoma. These results establish the usefulness of global expression platforms to identify novel tumor markers.

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