Hisatsugu Mouri

Expression of Clusterin in Human Pancreatic Cancer

Introduction Clusterin, also known as apolipoprotein J, has been implicated in numerous processes, including active cell death. Clusterin is reported to be overexpressed in breast and prostate cancers. However, its expression in pancreatic cancer is yet to be reported. Aim and Methodology To examine clusterin expression and apoptosis in 52 pancreatic tissues, including specimens from 33 cases of pancreatic cancer, by immunohistochemistry. Results Clusterin was expressed in 49% (16) of 33 cases of pancreatic cancer, 50% (13) of 26 cases of chronic pancreatitis, and 67% (4) of 6 cases of mucinous cystadenoma. It was not expressed in normal pancreas. Clusterin mRNA was also expressed in the pancreatic cancer cell lines. Clusterin was significantly more highly expressed at stage I and II (well-differentiated and moderately differentiated cancers). Clusterin and apoptosis were localized in the same cells in pancreatic cancer. However, clusterin expression was not significantly associated with apoptosis in any pancreatic disease. Clusterin-positive patients with pancreatic cancer survived significantly longer. Conclusion These results suggest that clusterin expression is induced in pancreatic cancer as well as chronic pancreatitis and that downregulation of clusterin may be involved in the progression of pancreatic cancer.

The EGFR Ligands Amphiregulin and Heparin-Binding EGF-like Growth Factor Promote Peritoneal Carcinomatosis in CXCR4-Expressing Gastric Cancer

Purpose: Peritoneal carcinomatosis, often associated with malignant ascites, is the most frequent cause of death in patients with advanced gastric cancer. We previously showed that the CXCR4/CXCL12 axis is involved in the development of peritoneal carcinomatosis from gastric cancer. Here, we investigated whether epidermal growth factor receptor (EGFR) ligands are also involved in the development of peritoneal carcinomatosis from gastric cancer. Experimental Design: The functional involvement of expression of the ErbB family of receptors and/or EGFR ligands was examined in CXCR4-expressing human gastric cancer cells and fibroblasts, clinical samples (primary tumors and ascites), and an animal model. Results: High concentration of the EGFR ligands amphiregulin and heparin-binding EGF-like growth factor (HB-EGF), as well as of CXCL12, were present in malignant ascites. Human gastric cancer cell lines and primary gastric tumors, with high potential to generate peritoneal carcinomatosis, expressed high levels of EGFR and CXCR4 mRNA and protein. Both amphiregulin and HB-EGF enhanced the proliferation, migration, and functional CXCR4 expression in highly CXCR4-expressing gastric cancer NUGC4 cells. Amphiregulin strongly enhanced the proliferation of NUGC4 cells, whereas HB-EGF markedly induced the migration of fibroblasts. Moreover, HB-EGF and CXCL12 together enhanced TNFα-converting enzyme (TACE)-dependent amphiregulin shedding from NUGC4 cells. In an experimental peritoneal carcinomatosis model in mice, cetuximab effectively reduced tumor growth and ascites formation. Conclusions: Our results strongly suggest that the EGFR ligands amphiregulin and HB-EGF play an important role, interacting with the CXCL12/CXCR4 axis, in the development of peritoneal carcinomatosis from gastric cancer, indicating that these two axes may be potential therapeutic targets for peritoneal carcinomatosis of gastric carcinoma. Clin Cancer Res; 17(11); 3619–30. ©2011 AACR.

Serum Levels of Pancreatitis-Associated Protein in Digestive Diseases with Special Reference to Gastrointestinal Cancers

The serum levels of pancreatitis-associatedprotein (PAP) were measured in 196 patients withdigestive diseases and 15 healthy subjects by anenzyme-linked immunosorbent assay. The serum PAP levelswere significantly elevated in the patients withgastric, colorectal, biliary tract, hepatocellular, orpancreatic cancers compared with the healthy subjects.After curative resection of the tumor, serum PAP levels were significantly decreased. The serumPAP levels were not related to clinicopathologicalfactors except for the tumor size of pancreatic cancer.There were some cases of PAP-positive andcarcinoembryonic antigen (CEA) or carbohydrate antigen (CA)19-9-negative gastric and colorectal cancers. The serumPAP levels were also significantly elevated in thepatients with acute pancreatitis compared with those in not only the healthy subjects but also thepatients with chronic pancreatitis. The peak PAP levelswere significantly correlated with the severity of acutepancreatitis and reflected the clinical healing of the disease. The peak of serum PAP wassignificantly delayed compared with those of otherpancreatic enzymes. These results suggest that theincrease of serum PAP levels in patients withgastrointestinal cancers reflects an ectopic expression of PAPin cancer cells and that increased serum levels of PAPin acute pancreatitis are correlated with the diseaseseverity and are prolonged than those of other pancreatic markers.

Expression of mesothelin mRNA in pure pancreatic juice from patients with pancreatic carcinoma, intraductal papillary mucinous neoplasm of the pancreas, and chronic pancreatitis.

Objectives: In the gene expression analysis of pancreatic carcinoma (PCa) using serial analysis of gene expression (SAGE) according to Ryu et al, the tag for the mesothelin mRNA transcript was present in 7 of 8 SAGE libraries derived from PCa but not in the 2 SAGE libraries derived from normal pancreatic duct epithelial cells. Mesothelin mRNA expression was confirmed with in situ hybridization in all 4 resected primary PCa tumors and with RT-PCR in 18 of 20 PCa cell lines, whereas mesothelin protein expression was confirmed with immunohistochemistry in all 60 resected primary PCa tissues by Argani et al. We evaluated mesothelin mRNA expression in pure pancreatic juice (PPJ) obtained from patients with PCa, chronic pancreatitis (CP), and intraductal papillary mucinous neoplasm (IPMN) of the pancreas. Methods: We evaluated mesothelin mRNA expression in the PPJ obtained from 21 patients with PCa, 22 with CP, and 11 with IPMN with reverse transcriptase PCR (RT-PCR). The PCR products were analyzed with agarose gel electrophoresis. DNase I treatment before RT-PCR and direct sequencing of the RT-PCR bands were performed for the analysis of the RT-PCR bands. Results: Two products, of 308 and 226 bp, were obtained with RT-PCR, and the 308-bp RT-PCR product was confirmed as being that derived from the genomic DNA by direct DNA sequencing. Mesothelin mRNA expression was discovered using RT-PCR in 11 (52%) of 21 patients with PCa, 5 (45%) of 11 with IPMN, and 3 (14%) of 22 with CP. Fishers exact test revealed significant differences between PCa and CP for mesothelin mRNA (P < 0.01). Moreover, the RT-PCR product (226 bp) of mesothelin mRNA in the PPJ samples with PCa was generally stronger than that in the PPJ samples with IPMN. Conclusion: Expression of mesothelin mRNA in PPJ was not strictly specific to PCa and was apt to be stronger in PCa than in IPMN. Quantitative detection of mesothelin mRNA in PPJ may have potential diagnostic implications for pancreatic tumors.

Diagnostic utility of aberrant methylation of tissue factor pathway inhibitor 2 in pure pancreatic juice for pancreatic carcinoma

The tissue factor pathway inhibitor 2 (TFPI‐2) is a Kunitz‐type serine proteinase inhibitor. Recently, the aberrant methylation of TFPI‐2 was detected frequently in pancreatic carcinoma (PCa) tissues but not in normal pancreatic tissues. We analyzed the aberrant methylation of TFPI‐2 in the pure pancreatic juice (PPJ) aspirated endoscopically from patients with various pancreatic diseases. Using the highly sensitive methylation‐specific polymerase chain reaction (MSP) and quantitative MSP (Q‐MSP) assay, we investigated the aberrant methylation of TFPI‐2 in nine human PCa cell lines and in the PPJ from patients with PCa, intraductal papillary mucinous neoplasms (IPMN) and chronic pancreatitis (CP). The incidence of aberrant TFPI‐2 methylation was seven (77.8%) of nine PCa cell lines by Q‐MSP. In cell lines, the expression of TFPI‐2 mRNA by quantitative reverse transcription–polymerase chain reaction showed an inverse correlation to the aberrant methylation of TFPI‐2. The incidence of aberrant TFPI‐2 methylation in the PPJ was 21 (58.3%) of 36 PCa patients, three (17.6%) of 17 IPMN and one (4.8%) of 21 CP by MSP assay. Using a suitable cut‐off value of 2.5 according to the receiver operating characteristic curve, the incidence of aberrant TFPI‐2 methylation in the PPJ by real‐time MSP was 18 (62.1%) of 29 PCa patients, one (5.1%) of 17 IPMN and three (14.3%) of 21 CP, respectively. The incidence of quantitative TFPI‐2 hypermethylation in the PPJ with PCa was significantly higher than that with IPMN (P < 0.001) or CP (P < 0.001). Moreover, the aberrant methylation rate of TFPI‐2 in the PPJ was 100%, as observed (6/6) in the PCa patients with liver metastasis, and 86.7% (26/30) in stages IVa + IVb of PCa by Q‐MSP assay. These results suggest that promoter methylation of TFPI‐2 in the PPJ may be a useful marker in the diagnosis and progression of PCa using an endoscopically feasible approach. (Cancer Sci 2006; 97: 1267–1273)

Pancreatic cancer associated with granulocyte-colony stimulating factor production confirmed by immunohistochemistry

We report an 83-year-old man with pancreatic body cancer of 4.5 cm in diameter. Peripheral leukocyte count was 15,700/microl and the serum concentration of granulocyte-colony stimulating factor (G-CSF) was 123 pg/ml (normal, 6.0-21.9 pg/ml) on admission. Furthermore, not only K-ras codon 12 (GGT --> GAT) but also p53 at codon 247 (CGG --> CCG) mutations were identified in the pancreatic juice aspirated endoscopically. We performed chemotherapy with two courses of 5-fluorouracil, pirarubicin hydrochloride, and mitomycin-C, resulting in no beneficial effect. After the second course the patient developed interstitial pneumonia, probably caused by anticancer drugs, and died 4 months after the tumor was detected. In the autopsy tissue, the tumor macroscopically occupied the pancreas body and was 7 x 6 x 5 cm in size. Histopathologic diagnosis of the tumor was poorly differentiated adenosquamous carcinoma. Immunohistochemical staining of the autopsy tissue showed that pancreatic cancer cells were positive for G-CSF. This is the first case report of G-CSF-positive pancreatic cancer confirmed by immunohistochemistry.

Superoxide dismutase is induced during rat pancreatic acinar cell injury.

Introduction Free radicals and their scavengers are supposed to be involved in pancreatitis. Aims To investigate the expression of superoxide dismutase (SOD) in rat pancreatic acinar cell injury. Methodology and Results As an in vivo model, male WBN/Kob rats were used. Chronic pancreatitis developed spontaneously at 12 weeks in this model and progressed thereafter, but acinar regeneration was recognized at 20 weeks. By semi-quantitative reverse transcription–polymerase chain reaction (RT-PCR), manganese SOD (MnSOD) mRNA expression peaked at 8 and 20 weeks, whereas copper/zinc SOD (CuZnSOD) mRNA expression peaked at 12 and 20 weeks. Immunohistochemistry confirmed the localization of SOD in acinar cells. Acinar cell apoptosis peaked at 12 and 20 weeks. In an in vitro study, MnSOD mRNA expression peaked at 2 hours after the addition of arginine to culture medium, whereas apoptosis was increased at 24 hours. Conclusion Thus, the induction of SOD around the onset and at the late stage of chronic pancreatitis in the WBN/Kob rats implies pancreatic ischemia and acinar remodeling, respectively. From the in vitro results, MnSOD expression might reflect a defensive mechanism of acinar cells against oxidative stress or pro-apoptotic stimuli.

Preproenkephalin hypermethylation in the pure pancreatic juice compared with p53 mutation in the diagnosis of pancreatic carcinoma

BackgroundAberrant methylation of CpG islands is a common mechanism for the dysregulation of tumor suppressor genes in a variety of human malignancies. Preproenkephalin ppENK) hypermethylation is recognized in 90% of pancreatic carcinoma (PCa) tissues, but not in normal pancreas. We analyzed ppENK hypermethylation in pure pancreatic juice (PPJ) in patients with PCa, intraductal papillary mucinous neoplasms (IPMN), and chronic pancreatitis (CP), and elucidated its usefulness as a marker in the diagnosis of PCa compared with p53 mutation.MethodsPPJ was collected endoscopically from 28 patients with PCa, 15 patients with IPMN, and 20 patients with CP. DNA was extracted from the supernatant and the sediment of PPJ. Methylation-specific polymerase chain reaction was performed for hypermethylation analysis of ppENK. In addition, single-strand conformation polymorphism and direct sequencing were performed simultaneously to identify p53 mutations.ResultsThe incidence of ppENK hypermethylation in the supernatant and/or the sediment of PPJ was 50% (14 of 28) in patients with PCa. In contrast, the incidence of ppENK hypermethylation was 26.7% (4 of 15) in patients with IPMN, and 5% (1 of 20) in patients with CP (P < 0.002). The incidence of p53 mutations in the PPJ was 42.9% (12 of 28) in patients with PCa and 0% (0 of 20) in patients with CP. Furthermore, the incidence of ppENK hypermethylation and/or p53 mutations in the PPJ was enhanced to 67.9% (19 of 28) in patients with PCa in the combination assay.ConclusionsThese results suggest that ppENK hypermethylation in PPJ is specific for cancer, and the combination assay with p53 enhances the genetic diagnosis of PCa.

Epidermoid cyst of the intrapancreatic accessory spleen producing CA19-9

We report a rare case of an epidermoid cyst in an accessory spleen at the pancreatic tail with producing CA19‐9. A 55‐year‐old female was admitted to our hospital, Cancer Research Institute, Kanazawa University, for close examination of a cystic lesion at the pancreatic tail and a high serum CA19‐9‐value (176 U/mL). There were almost no abdominal symptoms related to the cystic lesion. A cystic tumor approximately 3 cm in diameter and composed of multilocular cysts without a protruding portion of the inner surface was found at the pancreatic tail by ultrasound sonography, computed tomography, and magnetic resonance imaging. Endoscopic retrograde pancreatography revealed that the main pancreatic duct shifted at the pancreatic tail and there was no communication between the main pancreatic duct and cystic lesion. Based on a preoperative diagnosis of mucinous cystic tumor, distal pancreatectomy with splenectomy was performed. Histological findings suggested an epidermoid cyst (3.5 × 3.0 cm) originating from an intrapancreatic accessory spleen. Immunohistochemical analysis of CA19‐9 in the epidermoid cyst showed clear staining of the inner epithelium of the cyst and amorphous or hyalinous cystic contents. The serum CA19‐9 value was confirmed to decline to normal 2 months after resection. Physicians should not forget this disease during differential diagnosis related to pancreatic cystic lesions with elevated levels of serum tumor markers, such as CA19‐9 or carcinoembryonic antigen, although this disease is extremely rare.

Pancreatitis-Associated Protein Levels in Pancreatic Juice from Patients with Pancreatic Diseases

Background: Pancreatitis-associated protein (PAP), the acute-phase protein of the pancreas, is overexpressed in acute pancreatitis. Serum PAP levels were reported to be useful as an indicator of the severity, prognosis and healing of acute pancreatitis. Although PAP was originally identified in pancreatic juice, there has been no clinical report on PAP levels in pancreatic juice. This study was conducted to determine levels of PAP in pancreatic juice (PJ-PAP) in various human pancreatic diseases. Methods: PAP levels in endoscopically aspirated PJ were measured by enzyme-linked immunosorbent assay in 86 patients with pancreatic diseases. Results: 55% of 22 patients with pancreatic cancer (PC) and 25% of 49 patients with chronic pancreatitis (CP) were positive (>350 ng/ml) for PJ-PAP. PJ-PAP levels were significantly higher in PC than in CP, in which PJ-PAP was also significantly higher than in 15 control subjects. There was no significant correlation between PJ-PAP and serum PAP, and combination assay of serum PAP and/or PJ-PAP detected 80% of PC cases and 44% of CP cases. Conclusions: We have demonstrated that human PAP could be detected in pancreatic juice from patients with pancreatic diseases. Determination of PAP in pancreatic juice might be helpful for early detection of pancreatic injury.