Koushiro Ohtsubo

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.

Endosonographic evaluation of c-kit-positive gastrointestinal stromal tumor.

AbstractBackground: Endosonographic features of c-kit–positive gastrointestinal stromal tumors (GISTs) were compared with those of leiomyomas and schwannomas. Methods: Twenty-four patients with gastric mesenchymal tumors who underwent endoscopic ultrasonography (EUS) and surgical treatment were enrolled. GISTs were defined as c-kit (CD117)–positive tumors, leiomyomas as desmin-positive and c-kit–negative tumors, and schwannomas as S-100–positive and c-kit–negative tumors. Invasion to adjacent organs or more than 20 mitotic counts per 50 high power fields indicated malignancy. Results: There were 19 GISTs, three leiomyomas, and two schwannomas. All five malignant tumors were GISTs. A marginal halo was found in 12 of 19 GISTs and in both of the schwannomas, but not in any of the three leiomyomas. The echogenicities of GISTs were low but higher than that of the normal proper muscle layer, whereas those of leiomyomas and schwannomas were usually low. Lobulation of the tumor surface was documented only in GISTs, particularly in malignant ones. The tumor doubling time of a malignant GIST was 9.3 months, and that of six benign GISTs was 18.7 months (range = 10.7–28.0 months). Conclusion: Marginal halo and relatively higher echogenicity on EUS might suggest GIST. Marginal lobulation and a short doubling time may be signs of a malignant GIST.

K-ras mutations in duodenal aspirate without secretin stimulation for screening of pancreatic and biliary tract carcinoma.

K‐ras mutations at codon 12 (KRM) have been detected in over 80% of tissues and pure pancreatic juice (PPJ) samples from patients with pancreatic carcinoma (PCa) and are promising genetic tumor markers. Aspirating PPJ not only requires technical skill, but is also exhausting for patients. The authors attempted to evaluate whether the detection of KRM in the duodenal aspirate (DA) obtained immediately after endoscopic retrograde cholangiopancreatography (ERCP), an easier sample‐collecting method than collecting PPJ, could be useful for the diagnosis of PCa and biliary tract carcinoma (BTCa).

Abnormalities of tumor suppressor gene p16 in pancreatic carcinoma: immunohistochemical and genetic findings compared with clinicopathological parameters.

Background. Abnormalities of the tumor suppressor gene p16 have been reported in a variety of human tumors but are rare in pancreatic carcinoma except for cancer cell lines and xenografts. Their clinicopathological significance remains unknown. The purpose of this study was to examine immunohistochemical and genetic alterations of p16 in primary pancreatic carcinoma tissues and to investigate the relation between abnormalities of p16 and clinicopathological parameters to elucidate their clinicopathological significance. Methods. We investigated p16 expression in 60 pancreatic carcinoma cases by immunohistochemistry using a monoclonal antibody clone G175-405. In addition, we analyzed genetic alterations of the p16 gene using DNA extracted from microdissected tissue of pancreatic carcinoma, by polymerase chain reaction, nonradioisotopic single-strand conformation polymorphism (non-RI-SSCP), DNA sequencing, and hypermethylation analyses using restriction enzymes. We compared the abnormalities of p16 alterations with clinicopathological parameters to elucidate their significance. Results. On immunohistochemical study, staining for p16 protein was strongly positive in 22 (37%) of 60 pancreatic carcinoma cases, weakly positive in 24 (40%), and negative in 14 (23%). In contrast, p16 mutations were recognized in 9 (15%) of the 60 pancreatic carcinoma cases. The incidence of p16 mutations was 2 (9%) in 22 cases of pancreatic carcinoma with strongly positive staining, 4 (17%) in 24 with weakly positive staining, and 3 (21%) in 14 with negative staining. Hypermethylation of p16 was detected in the two pancreatic carcinoma cases with weakly positive staining, although homozygous deletions were not found in any case. There was no significant correlation between the expression of p16 protein and any of the clinicopathological parameters. However, there was a tendency for the tumor to be larger in patients with decreased expression of p16 protein than in those with normal expression levels. In contrast, the tumor was significantly larger and the survival period significantly shorter for patients with pancreatic carcinoma with p16 mutation or hypermethylation than for those with pancreatic carcinoma with an intact p16 gene (P ≪ 0.05). Conclusions. These findings suggest that p16 alterations may participate in the aggressiveness of pancreatic carcinoma.

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.

Usefulness of supernatant of pancreatic juice for genetic analysis of K-ras in diagnosis of pancreatic carcinoma.

Aims To ascertain whether analysis of K-ras mutations at codon 12 (KRM) in the supernatant of pure pancreatic juice (PPJ) is more useful for the diagnosis of pancreatic carcinoma (PCa) than that in sediment, the authors analyzed KRM in DNA extract from not only the sediment but also the supernatant of PPJ and compared the results. Methodology PPJ was collected endoscopically from 19 patients with PCa and 25 patients with chronic pancreatitis (CP). DNA was extracted from the supernatant and the sediment of PPJ. Mutant allele-specific amplification (MASA) was performed for KRM analysis with the DNA extracts from these samples. Results The incidence of KRM in the supernatant of PPJ was 89% (17 of 19) in patients with PCa and 28% (7 of 25) in patients with CP, whereas that in the sediment was 79% (15 of 19) in patients with PCa and 20% (5 of 25) in patients with CP. Although there was no significant difference in KRM incidence between supernatant and sediment, the positive rate of KRM was higher in the former. Additionally, with regard to the PCa cases, KRM were found in the supernatant alone in four cases and in the sediment alone in two cases. Consequently, by a combination assay, all of the patients with PCa showed KRM in either the supernatant or sediment of PPJ. Although there was no relation between the incidence of KRM in PPJ and the location and size of tumor, and clinical stage of carcinoma in the patients with PCa, two patients with clinical stage I disease showed KRM in the supernatant. Conclusion These results suggest that the positive rate of KRM in the supernatant is not lower than that in the sediment, and simultaneous analysis of KRM in the supernatant and sediment of PPJ enhances the genetic diagnosis of PCa.

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)

Bcl-2 expression related to altered p53 protein and its impact on the progression of human pancreatic carcinoma

Summaryp53 and Bcl-2 are two important factors related to apoptosis and tumorigenesis. In this study, a series of 52 cases of pancreatic carcinoma (PC) were investigated using an immunohistochemical assay to determine whether altered expression of Bcl-2 and p53 has an impact on the progression of this malignancy. Cytoplasmic immunoreactivity for Bcl-2 and nuclear staining of p53 was found in 12 (23.1%) and 32 (63.5%) cases of PC respectively. Furthermore, an inverse correlation between the expression of p53 and Bcl-2 existed in this series (P < 0.01). In a subgroup, the proportion of tumours showing that p53-positive and Bcl-2-negative staining was increased with increasing histological grade and clinical stage (P < 0.05), and moreover, the survival period of those patients whose tumour had this staining was shorter than those with other staining patterns of combined p53 and Bcl-2 (P < 0.05). Therefore, it is concluded that simultaneously aberrant expression of Bcl-2 and p53 may confer PC with more malignant clinicopathological characteristics.