Ayman Rahman

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 ∼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 (β 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.

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.

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.

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 immuneresponsiveness in certain instances.

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 pre-operative 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 non-malignant periampullary diseases and 204 ± 65 ng/ml in age-matched healthy controls (P

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.

A Subset of Pancreatic Adenocarcinomas Demonstrates Coamplification of Topoisomerase IIα and HER2/neu Use of Immunolabeling and Multicolor FISH for Potential Patient Screening and Treatment

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.

MAGE1 Is Expressed by a Subset of Pancreatic Endocrine Neoplasms and Associated Lymph Node and Liver Metastases

AbstractBackground.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 (minimally detectable expression), 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.

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.