Yuichi Sanada

Histopathologic evaluation of stepwise progression of pancreatic carcinoma with immunohistochemical analysis of gastric epithelial transcription factor SOX2: comparison of expression patterns between invasive components and cancerous or nonneoplastic intraductal components.

Objectives: The purpose of this study was to perform histopathologic and immunohistochemical analyses of gastric transcription factor SOX2 and gastric mucin MUC5AC to better understand the stepwise progression of pancreatic carcinoma. Methods: Twenty-eight representative sections from 14 surgically resected pancreatic carcinomas were assessed microscopically. Sites of pancreatic intraepithelial neoplasia (PanIN) were counted, and histologic subtypes of invasive ductal carcinoma (IDC) were determined. The expression of SOX2 and MUC5AC in PanIN and IDC was examined immunohistochemically. Results: One hundred thirty-eight PanINs were identified. In 4 of the 14 cases, gradual transition from PanIN-1A to PanIN-3 was observed in a single duct, suggesting stepwise progression. The expression of MUC5AC increased with the progression of lesions from PanIN-1A to PanIN-3. SOX2 was expressed in only 6 of 107 early PanINs (5.8%). Out of 31 PanIN-3s, 7 were positive (22.6%), and SOX2 protein was localized in the nuclei of cells of the basal epithelium or in the vicinity of luminal necrosis. In addition, SOX2 was frequently and strongly expressed in poorly differentiated (57.1%) and neurally invasive (63.6%) components. Conclusions: The results of our histopathologic examinations suggest that PanIN progresses stepwise to IDC. Immunohistochemistry results suggest that SOX2 is involved in later events of carcinogenesis.

Down-regulation of the claudin-18 gene, identified through serial analysis of gene expression data analysis, in gastric cancer with an intestinal phenotype.

Gastric cancer (GC) is one of the most common malignancies worldwide. Genes whose expression is down‐regulated in GC may be tumour suppressor genes. In the present study, genes with decreased expression in GC were screened for by serial analysis of gene expression (SAGE) data analysis and reverse transcription (RT)‐polymerase chain reaction (PCR), and CLDN18 (encoding claudin‐18) was identified. Quantitative RT‐PCR revealed that expression of CLDN18 was down‐regulated in 13 (56.5%) of 23 GCs. Immunostaining showed that normal gastric mucosa and Paneth cells of the duodenum expressed claudin‐18 on cell membranes. Expression of claudin‐18 was reduced in several intestinal metaplasias of the stomach. Of 20 samples of gastric adenoma, 18 (90.0%) showed decreased claudin‐18 expression. Down‐regulation of claudin‐18 was observed in 84 of 146 GCs (57.5%) and correlated with poor survival in 65 advanced GCs (p = 0.0346). In addition, expression of the gastric and intestinal phenotypes of GC was examined by immunostaining for MUC5AC, MUC6, MUC2, and CD10. Of 38 GCs showing only the intestinal phenotype, down‐regulation of claudin‐18 was observed in 28 (73.7%), whereas in the remaining 108 GC cases, down‐regulation of claudin‐18 was observed in 56 (51.9%) (p = 0.0224). These results indicate that claudin‐18 is a good marker of poor survival in GC. Down‐regulation of claudin‐18 may be involved in GCs with an intestinal phenotype, and may be an early event in gastric carcinogenesis. Copyright

Differential expression of claudin-2 in normal human tissues and gastrointestinal carcinomas

Claudins are involved in the formation of tight junctions in epithelial and endothelial cells. Claudins form a family of 24 members displaying organ- and tissue-specific patterns of expression. In the present study, we evaluated the specificity of the claudin-2 expression in various normal human tissues and gastrointestinal cancers by quantitative reverse transcriptase–polymerase chain reaction and immunohistochemistry. In 14 various normal tissues, claudin-2 mRNA was expressed in the kidney, liver, pancreas, stomach, and small intestine; the highest level of which was detected in the kidney. Colorectal cancers (CRCs) expressed claudin-2 mRNA at high levels. Immunohistochemical analysis of claudin-2 in 146 gastric cancers (GCs) and 99 CRCs demonstrated claudin-2 expression in 2.1% of GCs and 25.3% of CRCs, respectively. There was no obvious correlation between claudin-2 expression and clinicopathological parameters of CRCs. These results suggest that the expression of claudin-2 may involve organ specificity, and increased expression of claudin-2 may participate in colorectal carcinogenesis.

Serial analysis of gene expression of esophageal squamous cell carcinoma: ADAMTS16 is upregulated in esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies worldwide. To identify potential diagnostic markers for ESCC and therapeutic targets for ESCC, we used Serial Analysis of Gene Expression (SAGE) on one ESCC sample. We obtained a total of 14 430 tags, including 5765 that were unique. By comparing SAGE tags from the ESCC sample with those from normal human squamous esophagus, we found several genes that were differentially expressed between ESCC and normal squamous esophagus. Among these, we focused on the ADAM metallopeptidase with thrombospondin type 1 motif, 16 (ADAMTS16) gene because quantitative RT‐PCR analysis showed a high level of ADAMTS16 expression in eight out of 20 ESCC samples (40%), but not in 15 kinds of normal tissues. Western blot analysis also showed upregulation of ADAMTS16 protein in ESCC tissues. Furthermore, ADAMTS16 protein was detected in culture media from the TE5 esophageal cancer cell line. Knockdown of ADAMTS16 in TE5 cells inhibited both cell growth and invasion ability. Our present SAGE data provide a list of genes potentially associated with ESCC. ADAMTS16 could be a novel diagnostic and therapeutic target for ESCC.

Contribution of thymidylate synthase to gemcitabine therapy for advanced pancreatic cancer.

Objectives: Thymidylate synthase (TS) inhibitors activate human equilibrative nucleoside transporter 1. We evaluated the contribution of TS expression to determine a treatment method providing an effect from gemcitabine (GEM). Methods: The expression of 5-fluorouracil (5-FU) and GEM metabolic factors (5-FU: TS, dihydropyrimidine dehydrogenase, orotate phosphoribosyltransferase; GEM: human equilibrative nucleoside transporter 1, deoxycytidine kinase, cytidine deaminase, 5&vprime;-nucleotidase) were studied in 7 pancreatic cancer cell lines by Western blotting, and drug resistance was evaluated by 3-[4,5-dimethylthiazol]-2,5-dephenyl tetrazolium bromide assay. The expression of 5-FU factors was observed immunohistochemically in resected pancreatic cancer specimens. Results: Gemcitabine concentrations that inhibited colony formation by 50% correlated with TS protein expression (P = 0.0169). With a 5-FU non-growth-inhibiting dose, GEM concentrations that inhibited colony formation by 50% were significantly reduced by one fourth to one tenth. Knockout of TS expression by small interfering RNA decreased resistance to GEM in the cell lines (P = 0.0019). Immunohistochemically, TS expression related to disease-free survival time of patients treated with GEM (P = 0.0224). A high expression of 5-FU factors was detected: orotate phosphoribosyltransferase: differentiated cases (P = 0.0137), lower T factor (P = 0.0411); dihydropyrimidine dehydrogenase: nerve invasion (P = 0.0188), lymph node recurrence (P = 0.0253); TS, positive N factor (P = 0.0061). Conclusions: The expression of TS provides an alternative source of substrate for DNA synthesis and positively correlates with GEM resistance and shortened patient survival. Abbreviations: GEM - gemcitabine, 5-FU - 5-fluorouracil, TS - thymidylate synthase, DPD - dihydropyrimidine dehydrogenase, OPRT - orotate phosphoribosyltransferase, hENT1 - human equilibrative nucleoside transporter 1

Expression of orotate phosphoribosyltransferase (OPRT) in hepatobiliary and pancreatic carcinoma.

The purpose of this study was to clarify the role of orotate phosphoribosyltransferase (OPRT) in the progression of hepatobiliary and pancreatic carcinomas. Representative sections from 8 surgically resected pancreatic carcinomas, 5 gallbladder carcinomas and 19 hepatocellular carcinomas (HCCs) were examined microscopically. Sites of pancreatic intraepithelial neoplasia (PanIN) were counted, and histologic subtypes of invasive ductal carcinoma of the pancreas (IDC) were determined. Gallbladder carcinomas and HCCs were examined histologically, and the subtypes and spread patterns were assessed. Expression of OPRT was examined immunohistochemically. A total of 75 PanINs were identified. Expression of OPRT increased as lesions progressed from early to high-grade PanINs (PanIN-1A and -1B versus PanIN-2 and -3, p=0.0004). Three (37.5%) of the 8 pancreatic IDCs were positive for OPRT. In the remaining 5 cases, OPRT was expressed only in the neoplastic ducts adjacent to PanIN-3s. In gallbladder carcinomas, mucosal neoplastic epithelium showed dense cytoplasmic expression in 4 of the 5 cases, but expression was absent in the deeply invasive lesions. Among HCCs, 15 of the 19 cases were negative for OPRT in the central area of the tumor, but 8 of the 19 cases expressed OPRT in vascularly invasive lesions. Our data suggest that OPRT is involved in early events of pancreatic and gallbladder carcinogenesis and invasion of HCC.

Liver Abscess after Common Hepatic Artery Embolization for Delayed Hemorrhage Following Pancreaticoduodenectomy: A Case Report

A 55-year-old man underwent pancreaticoduodenectomy for bile duct carcinoma in March 2009. The patient developed anastomotic leakage and had a short episode of hemorrhage from the drainage tubes with spontaneous disappearance. CT and upper endoscopy did not reveal the source of bleeding. A massive life-threatening hemorrhage occurred on the 18th postsurgical day. Emergency angiography showed a 2.7-cm pseudoaneurysm of the gastroduodenal artery stump, and hepatic artery embolization was performed. After embolization, an abscess appeared in segments 2/3 of the liver without involving the right lobe. We treated conservatively by drainage and antibiotics. During the course of therapy after embolization, the patient experienced several episodes of high fever but did not develop hepatic failure. On the 68th day after embolization, the abscess had penetrated to the lesser sac, which was immediately treated by percutaneous drainage. Anastomotic leakage was treated by continuous irrigation from the drain, for which complete resolution was achieved by the 34th day after embolization. The patient was discharged 101 days after embolization. Imaging and the clinical course demonstrate a unique mechanism of abscess formation after embolization.

Dihydropyrimidine dehydrogenase and orotate phosphoribosyltransferase in esophageal cancer patients: Correlation with clinicopathological factors and prognosis

Thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD) and orotate phosphoribosyltransferase (OPRT) are fluoropyrimidine metabolic enzymes which play important roles in the response of cancer patients to chemotherapy. In esophageal cancer, little is known about the relationship between the expression of these enzymes and corresponding clinicopathological features. In the present study, TS, DPD and OPRT expression levels were evaluated in 72 resected esophageal cancer specimens using immunohistochemistry. The relationship between enzyme expression and clinicopathological features was assessed using Fishers exact test or the χ2 test (categorical variables), or the Mann-Whitney rank-sum test (continuous variables). Survival curves were calculated using the Kaplan-Meier method, and differences evaluated using the log-rank test. The Cox proportional hazards model was also used. High DPD expression was associated with depth of invasion, nodal status, tumor stage, lymphatic invasion and venous invasion (P<0.001, P=0.004, P<0.001, P=0.006, P=0.038, respectively), as well as with decreased patient survival (P=0.007). In patients receiving adjuvant chemotherapy, low DPD expression did not significantly improve recurrence-free survival. OPRT was particularly expressed in esophageal cancer cells as compared to normal squamous cells. High OPRT expression was associated with depth of invasion and venous invasion (P=0.006, P=0.003, respectively). To conclude, in esophageal cancer DPD expression was associated with tumor progression and prognosis, and OPRT expression was correlated with carcinogenesis and tumor progression.

Successful Surgical Resection for Peritoneal Implantation of Hepatocellular Carcinoma at the Paracardial Portion

Peritoneal implantation from hepatocellular carcinoma has been rarely reported. It may occur at various sites. Here we present a surgically resected case of peritoneal implantation to the diaphragm from hepatocellular carcinoma. A 50-year-old woman underwent right hemihepatectomy extended to a medial part of Couinaud segment IV for hepatocellular carcinoma in May 2000. In December 2008, the elevation of alpha-phetoprotein and the appearance of a heterogeneously enhanced mass, with dimensions of 9 × 7 cm, and adjacent to the remnant liver and pericardium suggested intrahepatic recurrence with markedly enhanced growth. After transcatheter arterial embolization, surgical resection under laparotomy combined with median sternotomy was selected. Samples of pericardial fluid showed no malignancy after cytological examination. At the superior border of the tumor, the confluence of pericardium and diaphragm was displaced, but the tumor itself showed a generally expanding but not invasive growth. The resected tumor showed moderately differentiated hepatocellular carcinoma whose pathology revealed a peritoneal implantation to the diaphragm. The patient is in good health without any postoperative complications or any further sign of recurrence.