The risk of retinal detachment in high myopia after small incision coaxial phacoemulsification.

PURPOSE: Report incidence and risk of retinal detachment (RD)after coaxial phacoemulsification in high myopic patients. DESIGN: Retrospective consecutive interventional study. SETTING: Vissum-Instituto Oftalmologico de Alicante, Spain. PATIENT OR STUDY POPULATION: A total of 439 eyes from 274 high myopic patients with spherical equivalent (SE) >or= 6.00 diopters (D) or axial length >or= 26.00 mm. Mean age 62.2, axial length 27.88 +/- 2.11 mm (26.0 mm to 37.9 mm), SE -13.3 +/- 5.6 (-6.00 D to -46.0 D). MAIN OUTCOME MEASURES: Incidence of RD, vitreous loss, and neodymium yttrium aluminum garnet (Nd:YAG) capsulotomy were evaluated after surgery at one, three, six, and 12 months then annually with mean follow-up 61.5 +/- 29.6 months. RESULTS: Incidence of RD was 2.7% (12/439 eyes). In eyes with 50 years of age was 2.52% (nine eyes). Cumulative risk of RD development by Kaplan-Meier analysis in all patients was 0.47%, 0.71%, 1.71%, 2.59%, and 3.28% at three, six, 15, 48, and 63 months until 105 months, respectively. In eyes 50 years of age was 0.58% and 2.96% at six and 52 months until 118 months after cataract surgery, respectively. No significant correlation was found between the occurrence of RD and vitreous loss (P

Increased expression and processing of the Alzheimer amyloid precursor protein in pancreatic cancer may influence cellular proliferation.

Abnormal cleavage of amyloid precursor protein (APP) in the central nervous system has been linked to the development of Alzheimer's disease. Recent work has identified additional roles for APP in peripheral tissue, such as cellular proliferation and motility. APP undergoes proteolytic processing to release a soluble NH(2)-terminal ectodomain fragment (sAPP), an Abeta or p3 peptide, and cytosolic COOH-terminal fragments. We have identified the up-regulation of APP expression in pancreatic cancer cells both in vitro and in vivo. APP undergoes high levels of proteolytic processing in pancreatic cancer cells, and sAPP can be detected in collected medium in vitro. Inhibition of sAPP signaling reduces pancreatic cancer cell number via a reduction in cellular proliferation. We propose that APP may function to promote growth in pancreatic cancer cells via signaling through sAPP and may therefore represent a novel therapeutic target in pancreatic cancer.

A subset of pancreatic adenocarcinomas demonstrates coamplification of topoisomerase IIalpha and HER2/neu: use of immunolabeling and multicolor FISH for potential patient screening andtreatment.

We sought to identify the frequency of amplification of the topoisomerase IIalpha gene (TOP2A) in pancreatic cancer and determine the usefulness of TOP2A immunolabeling in screening for TOP2A and human epidermal growth factor receptor (HER)2/neu amplification. We examined 55 pancreatic adenocarcinoma specimens for TOP2A immunolabeling and identified TOP2A protein expression in all specimens with a nuclear labeling index (NLI; positive nuclei/total nuclei x 100) of 5% to 80%. Normal pancreatic ductal epithelium, proposed to give rise to pancreatic adenocarcinoma, did not demonstrate detectable TOP2A expression. In a subset of specimens selected for fluorescence in situ hybridization analysis of TOP2A and HER2/neu amplification using a recently developed multicolor probe, 7 of 8 lesions with an NLI of 25% or more demonstrated TOP2A amplification, in contrast with 2 of 14 lesions with a TOP2A NLI of less than 25%. In 8 of 9 TOP2A-amplified cases, coamplification of HER2/neu was present, suggesting a potential relationship between TOP2A and HER2/neu in pancreatic adenocarcinoma. We propose that TOP2A immunolabeling be used in conjunction with a newly developed multicolor probe to screen patients with pancreatic adenocarcinoma to determine the best potential therapeutic modalities, such as TOP2A inhibitors, trastuzumab, or both.

Met proto-oncogene and insulin-like growth factor binding protein 3 overexpression correlates with metastatic ability in well-differentiated pancreatic endocrine neoplasms.

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 approximately 33,000 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 factor-related molecules (IGFBP1, IGFBP3, and MET), developmental factors (TBX3 and MEIS2), cytoskeletal factors (beta 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 proto-oncogene (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.

Global expression analysis of well-differentiated pancreatic endocrine neoplasms using oligonucleotide microarrays.

PURPOSE: Pancreatic endocrine neoplasms (PENs) are rare, mostly well-differentiated endocrine neoplasms, whose biology has been poorly characterized. Global expression microarrays can document abnormal pathways that impact on tumorigenesis and disease progression. EXPERIMENTAL DESIGN: RNA was extracted from eight well-differentiated PENs and three highly enriched pancreatic islet cell samples (80-90% purity), and examined using the Affymetrix U133A oligonucleotide microarray. Microarray data were normalized using dCHIP for identification of differentially expressed genes. PEN tissue microarrays were constructed from 53 archival PENs for immunohistochemical validation of microarray data. RESULTS: Sixty-six transcripts were overexpressed > or =3-fold in PENs compared with normal islet cells, including putative oncogenes (MLLT10/AF10), growth factors [insulin-like growth factor-binding protein 3 (IGFBP3)], cell adhesion and migration molecules (fibronectin), and endothelial elements (MUC18/MelCAM and CD31). A total of 119 transcripts were underexpressed < or =3-fold in PENs compared with normal islet cells, including cell cycle checkpoint proteins (p21/Cip1), the MIC2 (CD99) cell surface glycoprotein, putative metastasis suppressor genes (NME3), and junD, a MEN1-regulated transcription factor. Using PEN tissue microarrays, we confirmed the differential up-regulation of IGFBP3 (70%) and fibronectin (22%) and differential down-regulation of p21 (46%) and MIC2 (CD99; 91%) in PENs versus normal pancreatic islets. IGFBP3 overexpression was significantly more common in metastatic (93%) versus primary PEN lesions (60%), P=0.022. Fibronectin overexpression demonstrated a trend toward significance in lymphatic PEN metastases (55%) compared with primary PEN lesions (24%; P=0.14). CONCLUSIONS: Global expression analysis provides insight into tumorigenic pathways in PENs and may identify potential prognostic and therapeutic markers for these uncommon neoplasms.

Immunohistochemistry and in situ hybridization in pancreatic neoplasia.

There are many types of pancreatic neoplasms. Pathologic examination, which includes both routine (e.g., hematoxylin-and-eosin staining) and ancillary (e.g., immunohistochemistry and in situ hybridization) techniques, is essential in correctly typing a pancreatic neoplasm. This chapter focuses on the use of immunohistochemistry and in situ hybridization in the differentiation of pancreatic neoplasms. The materials and methods of these two techniques are described in detail.

HLA-G upregulation in pre-malignant and malignant lesions of the gastrointestinal tract.

HLA-G belongs to the nonclassical MHC class Ib group of molecules and has been implicated in mediating immune-responsiveness in various cancerous and non-cancerous cell types. We have examined HLA-G expression in a number of human gastrointestinal malignancies, including pancreatic ductal adenocarcinoma, ampullary cancer, biliary cancer, and colorectal cancer by immunolabeling analysis. We used indices of <5% (negative), 6-25%, 26-50%, 51-75%, and >75% (diffuse) to subclassify lesions based on percentage of positive cell labeling. Across all cancer subtypes, 52-79% of lesions demonstrated expression of HLA-G, with up to 33% of lesions demonstrating diffuse (>75%) expression. In addition, we utilized the neoplastic progression model of colorectal cancer to evaluate HLA-G protein expression in normal colon, tubulovillous adenomas, invasive cancer, and liver metastases arising from colorectal cancer. Focal HLA-G expression was detected in regions of normal colon adjacent to sites of adenomatous and cancerous lesions, as well as in all stages of cancer progression. Overall, the percentage of diffusely (>75%) labeled lesions appeared increased in preneoplastic and neoplastic conditions, as compared to normal colon. Specifically, tubulovillous adnenomas demonstrated pronounced diffuse labeling in 58% of lesions examined. No correlation with HLA-G expression and CD4+ or CD8+ T cells was identified. We propose that HLA-G expression is upregulated in a large percentage of gastrointestinal lesions and may serve to mediate immune-responsiveness in certain instances.

Evaluation of osteopontin as biomarker for pancreatic adenocarcinoma.

OBJECTIVES: Pancreatic adenocarcinoma is a deadly disease with an overall 5-year patient survival of less than 5%. This dismal prognosis of pancreatic cancer is largely due to the advanced stage of the disease at presentation. If pancreatic cancer could be diagnosed more readily and accurately using serum markers, patient survival could theoretically be improved by enabling more patients to avail of surgical resection. One candidate tumor marker recently identified by global gene expression analysis of pancreatic cancer is the secreted glycophosphoprotein osteopontin (OPN). In this study, we evaluate OPN as a serum marker of pancreatic adenocarcinoma. METHODS: In situ hybridization for OPN was performed on a pancreatic adenocarcinoma tissue microarray. Serum OPN levels were determined in preoperative sera from 50 patients with pancreatic cancer and 22 healthy control individuals by competitive ELISA. RESULTS: In situ hybridization for OPN performed on a tissue microarray revealed strong OPN mRNA signal in tumor-infiltrating macrophages in 8 of 14 pancreatic adenocarcinomas. In contrast, OPN expression was not seen in the pancreatic cancer cells themselves, nor was it seen in normal pancreatic tissue or in the macrophages distant from the infiltrating cancer. Serum OPN levels, as measured by ELISA, were elevated in the sera of 50 patients with resectable pancreatic adenocarcinoma compared to 22 healthy control individuals (mean +/- SD for OPN was 482 +/- 170 ng/ml and 204 +/- 65 ng/ml, respectively; P < 0.001). Using a cutoff level of 2 SD above the mean for healthy individuals, elevated OPN had sensitivity of 80% and specificity of 97% for pancreatic cancer. In contrast, only 62% of these patients with resectable pancreatic cancer had elevated CA19-9. CONCLUSIONS: Serum OPN may have utility as a diagnostic marker in patients with pancreatic cancer.

Gene expression profiling identifies markers of ampullary adenocarcinoma.

Ampullary adenocarcinoma is an aggressive cancer with a poor prognosis. Without surgical resection, ampullary adenocarcinomas can be difficult to distinguish from ampullary adenomas. The aim of this study was to identify differentially expressed genes in ampullary adenocarcinoma in order to identify candidate markers of the disease. The Affymetrix Human Genome U133 GeneChip set (HG-U133A and HG-U133B) was used to obtain gene expression profiles of 5 ampullary adenocarcinomas and 10 normal duodenal samples. Using fold change analysis we identified 235 fragments expressed at least fivefold higher in ampullary cancers than in normal duodenum. The expression profiles of eight candidate overexpressed genes (osteopontin, mesothelin, tissue inhibitor of metalloproteinases 1, mucin-1, mucin-5, fascin, heat shock protein 47, fibronectin 1) were confirmed by immunohistochemistry or in situ hybridization on tissue microarrays (TMA) containing 54 ampullary adenocarcinomas. One of these genes, osteopontin, was expressed 27-fold higher levels in ampullary adenocarcinomas compared to normal duodenum by genechip analysis. We therefore determined serum osteopontin levels in patients with ampullary neoplasms, patients with other periampullary diseases and in normal controls. Mean preoperative serum osteopontin levels as measured by competitive ELISA were 906 +/- 268 ng/ml in patients with ampullary cancer, 867 +/- 160 ng/ml in patients with an ampullary adenoma, 327.1 +/- 195.6 ng/ml in patients with nonmalignant periampullary diseases and 204 +/- 65 ng/ml in age-matched healthy controls (p < 0.001). Measurement of markers of ampullary cancer such as osteopontin may aid in the early detection and differential diagnosis of patients with periampullary lesions.

Analysis of novel tumor markers in pancreatic and biliary carcinomas using tissue microarrays.

Using global gene expression analyses, multiple novel tumor markers overexpressed in infiltrating ductal adenocarcinomas of the pancreas have recently been identified. However, the expression of these markers in morphologically similar adenocarcinomas of the biliary tree has not been investigated. The purpose of the present study was 3-fold. First, we used 8 markers that have been shown to be overexpressed in whole tissue sections of pancreatic adenocarcinomas to validate tissue microarrays (TMAs) created from a series of pancreatic adenocarcinomas (n=68). The labeling patterns of 6 epithelial markers (fascin, mucin 4, 14-3-3sigma, prostate stem cell antigen, topoisomerase IIalpha, and cdc2/p34) were concordant with previously published studies on whole tissue sections, yet required far fewer slides and reagents. Mesothelin, an epithelial marker, and heat shock protein 47, a marker of peritumoral desmoplasia, showed lower levels of expression in the TMAs when compared with whole tissue sections. Second, we examined the previously unknown expression of the same 8 novel tumor proteins in cancers of the biliary tree by using TMAs created from a series of intrahepatic cholangiocarcinomas, gallbladder adenocarcinomas, and adenocarcinomas of the distal common bile duct (n=38). Each of the 8 markers was overexpressed in the biliary cancers, ranging from 14% demonstrating at least focal labeling with prostate stem cell antigen to 100% labeling with cdc2/p34. Most of the markers showed lower frequencies of expression in the biliary tract carcinomas in comparison to the pancreatic adenocarcinomas. In addition, expression patterns varied with location in the biliary system (intrahepatic versus gallbladder versus distal common bile duct). These differences were statistically significant (P<0.05) for mesothelin, mucin 4, and heat shock protein 47. Finally, the expression of selected markers in neoplastic progression of gallbladder cancer was examined. Two markers, fascin and mesothelin, showed up-regulation of expression with transition from carcinoma in situ to invasive adenocarcinoma, implicating a role for these markers in neoplastic progression. The results of this study indicate that TMA technology provides valid and cost-effective means to screen large numbers of novel tumor markers, even in tumors such as pancreatic and biliary adenocarcinomas that characteristically have abundant desmoplastic stroma. In addition, novel tumor markers of pancreatic adenocarcinomas show similar, yet not identical, expression patterns in biliary carcinomas. Therefore, these markers are potentially useful in developing diagnostic tests and treatment paradigms for tumors involving the biliary system.

Loss of p27 nuclear expression in a prognostically favorable subset of well-differentiated pancreatic endocrine neoplasms.

Decreased expression of p27 occurs in aggressive colon, breast, and prostate neoplasms; p27 loss often correlates with worsened prognosis. Paradoxical overexpression has been described in benign and malignant pancreatic endocrine neoplasms (PENs). To investigate prognostic usefulness of p27 expression in PENs, we immunolabeled 42 primary PENs, with or without metastases, for p27 and separated lesions using a nuclear labeling index (NLI) of 10%. Of the 42 lesions, 26 demonstrated a 10% or higher NLI and 16 an NLI less than 10%. Comparison of lymph node status revealed that 50% of primary PENs with a 10% or higher NLI (13/26) demonstrated lymph node metastases, whereas only 6% of lesions with an NLI of less than 10% (1/16) demonstrated lymph node metastases (P = .0067). We next examined 11 liver and 7 lymph node metastases for p27 immunolabeling to determine whether p27 also is paradoxically retained in lesions that have metastasized. All 18 lesions demonstrated an NLI of 10% or higher for p27. Expression of p27 protein therefore appears to be lost in a subset of well-differentiated PENs with indolent features but paradoxically retained in PENs associated with metastatic disease.

Cyclooxygenase 2 expression in pancreatic adenocarcinoma and pancreatic intraepithelial neoplasia: an immunohistochemical analysis with automated cellular imaging.

We immunohistochemically examined material from 36 pancreata (adenocarcinomas, 30 lesions; pancreatic intraepithelial neoplasia [PanIN], 65; normal pancreatic ducts, 30) for cyclooxygenase 2 (COX-2) with an automated platform. We analyzed 7 to 10 discrete foci and generated an average percentage of positive cells and average staining intensity for each lesion. These 2 values were then multiplied to create an overall "HistoScore" for each lesion. COX-2 demonstrated considerable heterogeneity of expression between and within cases. The overall average percentage of positive cells in adenocarcinomas was 47.3%; in PanINs, 36.3%; and in normal ducts, 19.2%. COX-2 was expressed in more than 20% of cells in 23 adenocarcinomas (77%), 42 PanINs (65%), and 12 normal ducts (40%). The overall average HistoScore for adenocarcinomas was 6.1; for PanINs, 5.4; and for normal ducts, 3.5. Significant differences in COX-2 expression were demonstrable in adenocarcinomas vs normal ducts, PanINs vs normal ducts, and PanIN 2/3 vs PanIN 1a/1b. In general, the pattern of COX-2 expression increased from normal to PanIN to adenocarcinoma. The up-regulation of COX-2 in a subset of noninvasive precursor lesions makes it a potential target for chemoprevention with selective COX-2 inhibitors.

Immunohistochemical validation of a novel epithelial and a novel stromal marker of pancreatic ductal adenocarcinoma identified by global expression microarrays: sea urchin fascin homolog and heat shock protein 47.

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.

MAGE1 is expressed by a subset of pancreatic endocrine neoplasms and associated lymph node and liver metastases.

BACKGROUND: MAGE1 was originally isolated from human melanoma cells as a target antigen for autologous cytotoxic T lymphocytes. Expression of MAGE1 has subsequently been identified in a number of neoplastic cell types, including testicular germ cell and breast cancer cells, which has led to the development of antitumor MAGE1 vaccines. AIM OF THE STUDY: To determine if Mage-1 is expressed in pancreatic endocrine neoplasms (PENs) and PEN metastases. METHODS: We utilized immunolabeling analysis for Mage-1 on 49 primary PENs, 11 liver metastases, and 6 lymph node metastases. A semiquantitative labeling index (LI) of 0 (no expression), 1, 2 (moderate expression), and 3 (intense expression, correlating with internal control markers) was used to determine relative amounts of MAGE1 expression in these lesions. RESULTS: We have identified MAGE1 expression in a subset (42 of 49; 86%) of PENs. Normal pancreatic ducts, present in tissue adjacent to PENs, were utilized as a positive control for Mage-1 immunolabeling (index score 3); no other detectable labeling for Mage-1 was evident in normal pancreatic tissue. Primary PENs, with or without metastases (mean LI score 1.2 vs 1.0, respectively), did not demonstrate a significant difference in Mage-1 LI, although intratumoral heterogeneity was apparent in some, but not all, of these lesions. Lymph node metastases (mean score 2.0) demonstrated a significant increase in Mage-1 LI as compared to primary, non-metastatic lesions (p = 0.04984) and primary metastatic lesions (p = 0.02351). In contrast, six patients with a survival of less than one year demonstrated a low Mage-1 LI (mean score, 0.58). CONCLUSIONS: MAGE1 expression is present in a subset of primary PENs and in lymph node metastases, and may therefore serve as a useful marker and potential therapeutic target in PENs. Furthermore, the absence of Mage-1 expression in a subset of primary PENs may indicate a worsened prognosis.

Inhibitory role of IFN-gamma-inducible lysosomal thiol reductase in T cell activation.

IFN-gamma-inducible lysosomal thiol reductase (GILT) is a unique thiol reductase with optimal enzymatic activity at low pH. GILT plays a crucial role in unfolding the antigenic proteins in preparation for their proteolytic cleavage and presentation of resulting peptides by MHC class II. In this study, we demonstrate that GILT is expressed in T lymphocytes and that it has an APC-nonrelated role in the regulation of T cell activation. Surprisingly, comparison of wild-type and GILT-deficient T cell activation in vitro revealed stronger responsiveness in the absence of GILT. The effect of GILT in reducing the proliferative and cytotoxic responses was endogenous to T cells and resulted from decreased sensitivity at the individual cell level. Therefore, a molecule with primarily lysosomal localization suppresses T cell activation, a process characterized by signal transmission from plasma membrane to cytoplasm and nucleus.

Liver metastases arising from well-differentiated pancreatic endocrine neoplasms demonstrate increased VEGF-C expression.

Pancreatic endocrine neoplasms (PENs) are uncommon, generally well-differentiated neoplasms that demonstrate prominent endocrine differentiation. Although the majority of PENs remain localized, malignant spread may occur via lymphatic or hematogenous routes. Angiogenic growth factors, including the vascular endothelial growth factor (VEGF) family, have been implicated in new vessel growth and hematogenous metastases, although this has not been studied in PENs. We therefore examined 19 primary well-differentiated PENs and 7 liver metastases to determine the expression of VEGF-A and its family member VEGF-C by immunolabeling analysis. VEGF-A immunoreactivity was evident only in scattered cells throughout all lesions. VEGF-C, however, demonstrated low-to-moderate expression in primary PENs by semiquantitative histoscore analysis (factor of labeling intensity by percentage of positive cells), with significantly increased expression in liver metastases (mean histoscore indices: primary PEN, 4.7 versus liver metastases, 9.5; Student's t test; P =.002773). Microvascular density of primary PENs and liver metastases did not appear to linearly correlate with VEGF-C expression. Examination of the VEGF-C-specific receptors VEGFR-2/KDR/Flk-1 and VEGFR-3/Flt-4 demonstrated intense endothelial immunoreactivity for VEGFR-2, as well as VEGFR-2 and -3 expression on the majority of neoplastic cells, suggesting a possible role in autocrine/paracrine neoplastic growth regulation. We postulate that the upregulation of VEGF-C may be involved in PEN progression and metastases, although not via a direct proangiogenic mechanism.

Identification of novel cellular targets in biliary tract cancers using global gene expression technology.

Biliary tract carcinoma carries a poor prognosis, and difficulties with clinical management in patients with advanced disease are often due to frequent late-stage diagnosis, lack of serum markers, and limited information regarding biliary tumor pathogenesis. RNA-based global analyses of gene expression have led to the identification of a large number of up-regulated genes in several cancer types. We have used the recently developed Affymetrix U133A gene expression microarrays containing nearly 22,000 unique transcripts to obtain global gene expression profiles from normal biliary epithelial scrapings (n = 5), surgically resected biliary carcinomas (n = 11), and biliary cancer cell lines (n = 9). Microarray hybridization data were normalized using dCHIP (http://www.dCHIP.org) to identify differentially up-regulated genes in primary biliary cancers and biliary cancer cell lines and their expression profiles was compared to that of normal epithelial scrapings using the dCHIP software as well as Significance Analysis of Microarrays or SAM (http://www-stat.stanford.edu/ approximately tibs/SAM/). Comparison of the dCHIP and SAM datasets revealed an overlapping list of 282 genes expressed at greater than threefold levels in the cancers compared to normal epithelium (t-test P <0.1 in dCHIP, and median false discovery rate <10 in SAM). Several pathways integral to tumorigenesis were up-regulated in the biliary cancers, including proliferation and cell cycle antigens (eg, cyclins D2 and E2, cdc2/p34, and geminin), transcription factors (eg, homeobox B7 and islet-1), growth factors and growth factor receptors (eg, hepatocyte growth factor, amphiregulin, and insulin-like growth factor 1 receptor), and enzymes modulating sensitivity to chemotherapeutic agents (eg, cystathionine beta synthase, dCMP deaminase, and CTP synthase). In addition, we identified several "pathway" genes that are rapidly emerging as novel therapeutic targets in cancer (eg, cytosolic phospholipase A2, an upstream target of the cyclooxygenase pathway, and ribosomal protein S6 kinase and eukaryotic translation initiation factor 4E, two important downstream mediators of the mitogenic Akt/mTOR signaling pathway). Overexpression of selected up-regulated genes was confirmed in tissue microarrays of biliary cancers by immunohistochemical analysis (n = 4) or in situ hybridization (n = 1), and in biliary cancer cell lines by reverse transcriptase PCR (n = 2). The majority of genes identified in the present study has not been previously reported in biliary cancers, and represent novel potential screening and therapeutic targets of this cancer type.