Dengfeng Cao

Differentially expressed genes in pancreatic ductal adenocarcinomas identified through serial analysis of gene expression.

Serial analysis of gene expression (SAGE) is a powerful tool for the discovery of novel tumor markers. The publicly available on-line SAGE libraries of normal and neoplastic tissues (http://www.ncbi.nlm.nih.gov/SAGE/) have recently been expanded; in addition, a more complete annotation of the human genome and better biocomputational techniques have substantially improved the assignment of differentially expressed SAGE “tags” to human genes. These improvements have provided us with an opportunity to re-evaluate global gene expression in pancreatic cancer using existing SAGE libraries. SAGE libraries generated from 6 pancreatic cancers were compared to SAGE libraries generated from 11 non-neoplastic tissues. Compared to normal tissue libraries, we identified 453 SAGE tags as differentially expressed in pancreatic cancer, including 395 that mapped to known genes and 58 “uncharacterized” tags. Of the 395 SAGE tags assigned to known genes, 223 were overexpressed in pancreatic cancer, and 172 were underexpressed. In order to map the 58 uncharacterized differentially expressed SAGE tags to genes, we used a newly developed resource called TAGmapper (http://tagmapper.ibioinformatics.org), to identify 16 additional differentially expressed genes. The differential expression of 7 genes, involved in multiple cellular processes such as signal transduction (MIC-1), differentiation (DMBT1 and Neugrin), immune response (CD74), inflammation (CXCL2), cell cycle (CEB1) and enzymatic activity (Kallikrein 6), was confirmed by either immunohistochemical labeling of tissue microarrays (Kallikrein 6, CD74 and DMBT1) or by RT-PCR (CEB1, Neugrin, MIC1, and CXCL2). Of note, Neugrin was one of the genes whose previously uncharacterized SAGE tag was correctly assigned using TAGmapper, validating the utility of this program. Novel differentially expressed genes in a cancer type can be identified by revisiting updated and expanded SAGE databases. TAGmapper should prove to be a powerful tool for the discovery of novel tumor markers through assignment of uncharacterized SAGE tags.

Expression of novel markers of pancreatic ductal adenocarcinoma in pancreatic nonductal neoplasms: additional evidence of different genetic pathways.

Solid pseudopapillary tumor, pancreatoblastoma, undifferentiated carcinoma with osteoclastic-like giant cells, and acinar cell carcinomas are rare pancreatic nonductal neoplasms. Compared to the significant advances in our understanding of the pathogenesis of pancreatic ductal adenocarcinomas in the last decades, the molecular mechanisms underlying pancreatic nonductal neoplasms are poorly understood. In order to elucidate their molecular pathogenesis, we constructed tissue microarrays to study the expression of some novel pancreatic ductal adenocarcinoma-associated tumor markers in these nonductal pancreatic neoplasms. We analyzed nine markers including tumor suppressor gene (14-3-3 sigma), proliferation marker (topoisomerase II alpha), epithelial markers (prostate stem cell antigen, mesothelin and cytokeratin 19), stromal markers (fascin, hsp47 and fibronectin), and gamma-synuclein whose function is not delineated. In addition, we included tumor suppressor gene DPC4 and oncogene Beta-catenin to further confirm their expression in pancreatic nonductal tumors. Our results showed that in contrast to pancreatic ductal adenocarcinomas that show loss of Dpc4 protein in 55% of cases, loss of Dpc4 expression is absent in pancreatic nonductal neoplasms. Expression of 14-3-3 sigma is frequently seen in both pancreatic nonductal neoplasms (25–100%) and ductal adenocarcinomas (89%). Aberrant nuclear expression of beta-catenin is common in pancreatic nonductal neoplasms, specifically in solid pseudopapillary tumors (88%) and pancreatoblastomas (100%) but is rarely seen in pancreatic ductal adenocarcinomas (<5%). Expression of topoisomerase II alpha is not seen in solid pseudopapillary tumors and undifferentiated carcinomas with osteoclastic-like giant cells but is focally seen in pancreatoblastomas (50%) and acinar cell carcinomas (85%). Expression of PSCA and mesothelin was observed in pancreatic nonductal neoplasms but their expression was seen less frequently (0–50%) and weaker than that in pancreatic ductal adenocarcinomas (60–100%). CK19, a marker of pancreatic ductal adenocarcinomas, is not expressed in pancreatic nonductal neoplasms. Expression of gamma-synuclein as well as stromal markers (fascin, hsp47 and fibronectin) is frequently seen in both. Our findings indicate pancreatic nonductal neoplasms have distinctive patterns of protein expression relative to pancreatic ductal adenocarcinomas and suggest that pancreatic nonductal neoplasms have different genetic pathways from the more common pancreatic ductal adenocarcinomas.

Prognostic significance of maspin in pancreatic ductal adenocarcinoma: tissue microarray analysis of 223 surgically resected cases

Maspin (SERPINB5), a serine proteinase inhibitor, was first identified as a potential tumor suppressor on the basis of its differential expression between normal mammary epithelial cells and human breast carcinoma cell lines. Recent studies have shown that maspin might be a prognostic tumor marker. Pancreatic ductal adenocarcinoma acquires maspin expression through hypomethylation of the maspin promoter. However, no study has investigated the prognostic significance of maspin expression in pancreatic ductal adenocarcinomas. In this study, we investigated maspin protein expression in a large series of 223 surgically resected pancreatic ductal adenocarcinomas using immunohistochemical staining and high throughput tissue microarrays. Maspin expression was correlated with postoperative survival and other clinicopathologic factors. Maspin was detected in 209 of these 223 (94% cases) pancreatic ductal adenocarcinomas including 39 (18% cases) focal (5–50% tumor cells) and 170 (76% cases) diffuse (>50% tumor cells). Fourteen (or 6% cases) pancreatic ductal adenocarcinomas did not show maspin expression by immunohistochemical staining (<5% tumor cells). Normal ductal epithelium is not labeled with maspin. Overexpression of maspin in pancreatic ductal adenocarcinoma is associated with worse postoperative survival especially in patients whose tumors exhibit diffuse expression of maspin. After adjusting other clinicopathologic factors, maspin expression remains to be an independent adverse prognosticator for postoperative survival. Maspin expression is not associated with patient age, gender, tumor size, tumor pathologic stage, lymph node status, and vascular invasion or perineural invasion. Nuclear labeling of maspin is associated with better tumor differentiation although this staining pattern is not associated with a better prognosis. In addition, maspin overexpression is also observed in 48% low-grade (grades 1a and 1b) pancreatic intraepithelial neoplasias (PanINs) and 78% high-grade (grades 2 and 3) PanINs, suggesting that maspin upregulation occurs early during the multi-step progression model of pancreatic ductal adenocarcinoma.

Identification of novel highly expressed genes in pancreatic ductal adenocarcinomas through a bioinformatics analysis of expressed sequence tags.

In most microarray experiments, a significant fraction of the differentially expressed mRNAs identified correspond to expressed sequence tags (ESTs) and are generally discarded from further analyses. We used careful bioinformatics analyses to characterize those ESTs that were found to be highly overexpressed in a series of pancreatic adenocarcinomas. cDNA was prepared from 60 non-neoplastic samples (normal pancreas [n = 20], normal colon [n = 10], or normal duodenal mucosal [n = 30]) and from 64 pancreatic cancers (resected cancers [n = 50] or cancer cell lines [n = 14]) and hybridized to the complete Affymetrix Human Genome U133 GeneChip® set (arrays U133A and B) for simultaneous analysis of 45,000 fragments corresponding to 33,000 known genes and 6,000 ESTs. The GeneExpress® software system Fold Change Analysis Tool was used and 60 ESTs were identified that were expressed at levels at least 3-fold greater in the pancreatic cancers as compared to normal tissues. Searches against the human genomic sequence and comparative genomic analysis of human and mouse genomes was carried out using basic local alignment search tools (BLAST), BLASTN, and BLASTX, for identifying protein coding genes corresponding to the ESTs. Subsequently, in order to pick the most relevant candidate genes for a more detailed analysis, we looked for domains/motifs in the open reading frames using SMART and Pfam programs. We were able to definitively map 43 of the 60 ESTs to known or novel genes, and 15 of the ESTs could be localized in close proximity to a gene in the human genome although we were unable to establish that the EST was indeed derived from those genes. The differential expression of a subset of genes was confirmed at the protein level by immunohistochemical labeling of tissue microarrays (inhibin b A [INHBA] and CD29) and/or at the transcript level by RT-PCR (INHBA, AKAP12, ELK3, FOXQ1, EIF5A2, and EFNA5). We conclude that bioinformatics tools can be used to characterize differentially overexpressed ESTs, and that some of these ESTs may represent diagnostically and therapeutically useful targets that might be missed using data solely from currently annotated databases.

Little or no residual prostate cancer at radical prostatectomy: Vanishing cancer or switched specimen? A microsatellite analysis of specimen identity

With more vigilant screening for prostate cancer, there has been an associated increase in patients with little or no residual cancer at radical prostatectomy after an initial diagnosis of minute cancer on needle biopsy. This raises a critical question as to whether the biopsy and subsequent radical prostatectomy in these patients are from the same patient. We used PCR-based microsatellite marker analysis to perform identity test in 46 men (35 with minute cancer and 11 with no residual cancer). Of them, 41 were interpretable, including 31 with minute cancer and 10 with no residual cancer. All 31 interpretable cases with minute cancer showed match between the initial biopsy and radical prostatectomy specimens. Nine of the 10 interpretable cases with no residual cancer showed match and 1 showed mismatch. The remaining 5 cases (4 with minute cancer and 1 with no residual cancer) were considered uninterpretable due to technical problems. The initial biopsy of the mismatched case had high-grade cancer (Gleason score 4 + 4 = 8) measuring 9.6 mm in length with perineural invasion. Our results confirm that, in most cases of “vanishing cancer” in radical prostatectomy specimens, it reflects a chance sampling of a minute cancer and not a switch in specimens. However, specimen switch can rarely occur, and if there is high grade or a lot of cancer on the biopsy with no or very minimal cancer in the radical prostatectomy specimen, one should evaluate for patient identity.

Positive immunohistochemical staining of KIT in solid-pseudopapillary neoplasms of the pancreas is not associated with KIT/PDGFRA mutations

Solid-pseudopapillary neoplasms of the pancreas are uncommon neoplasms of low malignant potential and of uncertain histogenesis. A small percentage of patients develop metastatic disease and some succumb to disease. The management of patients with metastatic disease or unresectable tumor, and patients who are just not good surgical candidates is problematic. Novel therapy targets are needed. Successful treatment of metastatic and unresectable gastrointestinal stromal tumors with KIT kinase inhibitor, imatinib mesylate (Gleevec), makes it intriguing to look at the status of KIT in solid-pseudopapillary neoplasms of the pancreas. In this study, we investigated KIT expression in 50 solid-pseudopapillary neoplasms by immunohistochemical staining. Of the 50 (50%) solid-pseudopapillary neoplasms, 25 showed diffuse expression (in >50% neoplastic cells) of KIT and additional five (10%) cases showed focal staining (in 10–50% neoplastic cells). Expression of KIT was not associated with tumor behavior and prognosis. A subset of 11 cases showing diffuse KIT expression detected by immunohistochemical staining were further evaluated for the presence of activating mutations in KIT exons 9, 11, 13 and 17, and PDGFRA exons 12 and 18 using PCR amplification followed by direct sequencing. However, no KIT or PDGFRA mutations were identified in any of these 11 cases tested, suggesting that the overexpression of KIT is probably not due to activating mutations in KIT or PDGFRA. The exact mechanism of KIT overexpression in solid-pseudopapillary neoplasms remains to be elucidated. One possible mechanism is gene dose effect (increased copies of KIT gene). Experience in gastrointestinal stromal tumors and other tumors have shown that mutation-mediated activation of KIT or PDGFRA is a prerequisite for clinical response with imatinib mesylate. Thus, lack of mutations in KIT or PDGFRA in solid-pseudopapillary neoplasms suggests that imatinib mesylate is less likely to be effective in the treatment for patients with metastatic disease or unresectable tumor, and patients who are just not good surgical candidates.

Aberrant expression of maspin in idiopathic inflammatory bowel disease is associated with disease activity and neoplastic transformation

AbstractBackground. Maspin is both overexpressed in tumors and inflammation, implicating a possible role in bridging inflammation and neoplasia. Idiopathic inflammatory bowel disease (IBD) and IBD-associated dysplasias and carcinomas represent a prototype for studying the relationship between chronic inflammatory states and neoplasia. Aim of Study. To investigate expression of maspin in IBD and IBD-associated dysplasia and colorectal carcinoma. Methods. Immunohistochemical labeling of maspin was examined using tissue microarrays constructed from archival biopsy and resection tissue from 90 patients with 125 histologically defined lesions including 30 with inactive chronic IBD, 51 with active chronic IBD, 4 IBD-associated foci with epithelial changes indefinite for dysplasia (IFD), 7 with IBD-associated low-grade epithelial dysplasia (LGD), 8 with IBD-associated high grade epithelial dysplasia (HGD), and 25 with IBD-associated invasive colorectal adenocarcinomas. Results. Maspin was expressed in 47/51 (92%) active chronic IBD lesions, which was significantly higher than both inactive chronic IBD (13/30, 43%) and normal mucosa (1 of 9, 11%) (p<0.01); in particular, the diffuse pattern of maspin expression was significantly higher in active IBD (41/51, 80%), compared with inactive IBD (5/30, 17%) and normal mucosa (0%) (p<0.01). In the multistage progression model of colitis-associated neoplasia, aberrant labeling was observed at the earliest stages, with 3/4 (75%) IFD foci, 6/7 (86%) LGD, and 8/8 (100%) HGD specimens expressing maspin, virtually always in a diffuse pattern. Expectedly, 22/25 (88%) of invasive IBD-associated cancers overexpressed maspin, including 21 with diffuse labeling. Conclusions. Maspin is significantly overexpressed in both active IBD and colitis-associated dysplasia compared to either inactive IBD or normal colonic mucosa, suggesting a potential role in disease “flare” as well as neoplastic progression. Targeting maspin for control of disease activity and cancer prophylaxis may be a promising novel therapeutic strategy for IBD.