Toshiyasu Iwao

Gene mutations of K-ras in gallbladder mucosae and gallbladder carcinoma with an anomalous junction of the pancreaticobiliary duct

ObjectiveIn this study, we examined the mutational spectrum of K-ras in cases of gallbladder and gallbladder carcinoma with an anomalous junction of the pancreaticobiliary duct (AJPBD).MethodsWe examined 35 gallbladders with AJPBD (20 with hyperplasia, 15 with carcinoma) and 38 gallbladders without AJPBD (four normal gallbladders, four with hyperplasia, six with adenoma, 24 with carcinoma). Polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) and direct sequencing were performed to detect mutations in codon 12 or 13 of K-ras.ResultsIn the cases with AJPBD, the prevalences of K-ras mutation were 15% (3/20) in hyperplasia, 60% (6/10) in stage I carcinoma, and 100% (5/5) in stage II–IV carcinoma. In the cases without AJPBD, the prevalences of K-ras mutation were 0% (0/4) in normal gallbladder, 0% (0/4) in hyperplasia, 17% (1/6) in adenoma, 7% (1/16) in stage I carcinoma, and 38% (3/8) in stage II–IV carcinoma. Prevalences of K-ras mutation in hyperplasia and carcinoma with AJPBD were greater than those without AJPBD (p < 0.05). The point mutation of GGT to GAT in codon 12 was frequently observed in the cases with AJPBD.ConclusionsThese results suggest that the specific K-ras mutation in codon 12 (GGT to GAT) may contribute to the early stage of carcinogenesis in the gallbladder with AJPBD.

Immunocytochemical detection of p53 protein from pancreatic duct brushings in patients with pancreatic carcinoma

It is often difficult to distinguish pancreatic carcinoma preoperatively from chronic pancreatitis. Therefore, we have developed a new method of detecting p53 immunoreactivity in cytologic material obtained by endoscopic retrograde pancreatic duct brushing (ERPDB).

Experimental trial for diagnosis of pancreatic ductal carcinoma based on gene expression profiles of pancreatic ductal cells.

Pancreatic ductal carcinoma (PDC) remains one of the most intractable human malignancies, mainly because of the lack of sensitive detection methods. Although gene expression profiling by DNA microarray analysis is a promising tool for the development of such detection systems, a simple comparison of pancreatic tissues may yield misleading data that reflect only differences in cellular composition. To directly compare PDC cells with normal pancreatic ductal cells, we purified MUC1‐positive epithelial cells from the pancreatic juices of 25 individuals with a normal pancreas and 24 patients with PDC. The gene expression profiles of these 49 specimens were determined with DNA microarrays containing >44 000 probe sets. Application of both Welchs analysis of variance and effect size‐based selection to the expression data resulted in the identification of 21 probe sets corresponding to 20 genes whose expression was highly associated with clinical diagnosis. Furthermore, correspondence analysis and 3‐D projection with these probe sets resulted in separation of the transcriptomes of pancreatic ductal cells into distinct but overlapping spaces corresponding to the two clinical classes. To establish an accurate transcriptome‐based diagnosis system for PDC, we applied supervised class prediction algorithms to our large data set. With the expression profiles of only five predictor genes, the weighted vote method diagnosed the class of samples with an accuracy of 81.6%. Microarray analysis with purified pancreatic ductal cells has thus provided a basis for the development of a sensitive method for the detection of PDC. (Cancer Sci 2005; 96: 387–393)

TP53 mutations in stage I gallbladder carcinoma with special attention to growth patterns

32 stage I cases of gallbladder carcinoma (GC) were examined to evaluate TP53 mutations with special attention to growth patterns. Their growth patterns were classified into two types: polypoid (P-type) and flat (F-type). 16 cases of GC were classified as P-type and 16 as F-type. p53 immunohistochemistry was performed using a mouse monoclonal anti-p53 antibody. Mutations in exons 5-8 were examined by polymerase chain reaction single strand conformation polymorphism (PCR-SSCP) and direct sequencing. The incidence of p53 immunoreactivity was greater in the cases of F-type (11/16, 69%) than those in P-type (14/16, 25%) (P < 0.05). PCR-SSCP or direct sequencing revealed that TP53 mutations were detected in all cases positive for p53 protein. These results suggest that TP53 mutations may contribute to the carcinogenesis of the F-type GC, and than this pathway in the F-type may differ from that in the P-type GC.

Screening of genes specifically activated in the pancreatic juice ductal cells from the patients with pancreatic ductal carcinoma.

Pancreatic ductal carcinoma (PDC) is one of the most intractable human malignancies. Surgical resection of PDC at curable stages is hampered by a lack of sensitive and reliable detection methods. Given that DNA microarray analysis allows the expression of thousands of genes to be monitored simultaneously, it offers a potentially suitable approach to the identification of molecular markers for the clinical diagnosis of PDC. However, a simple comparison between the transcriptomes of normal and cancerous pancreatic tissue is likely to yield misleading pseudopositive data that reflect mainly the different cellular compositions of the specimens. Indeed, a microarray comparison of normal and cancerous tissue identified the INSULIN gene as one of the genes whose expression was most specific to normal tissue. To eliminate such a “population‐shift” effect, the pancreatic ductal epithelial cells were purified by MUC1‐based affinity chromatography from pancreatic juice isolated from both healthy individuals and PDC patients. Analysis of these background‐matched samples with DNA microarrays representing 3456 human genes resulted in the identification of candidate genes for PDC‐specific markers, including those for AC133 and carcinoembryonic antigen‐related cell adhesion molecule 7 (CEACAM7). Specific expression of these genes in the ductal cells of the patients with PDC was confirmed by quantitative real‐time polymerase chain reaction analysis. Microarray analysis with purified pancreatic ductal cells has thus provided a basis for the development of a sensitive method for the detection of PDC that relies on pancreatic juice, which is routinely obtained in the clinical setting. (Cancer Sci 2003; 94: 263–270)

Pancreatic ductal carcinoma-specific genes identified by DNA microarray analysis with ductal cells

Pancreatic ductal carcinoma (PDC) remains the most intractable disorder amonggastroenterological malignancies, with a 5-year survival rate of <5%. Given the low 5-year survival rate even of individuals with small, resectable tumors, the sensitivity of current technologies is not sufficient to allow detection of pancreatic carcinoma at curable early stages. A cure for this disorder will thus depend on development of an approach that is able to detect tumors at an early stage of carcinogenesis. DNA microarray analysis allows the simultaneous monitor[ng of the expression of thousands of genes and is therefore a potentially suitable approach to identify PDC-specific genes. The high throughput of this methodology also may be disadvantageous, however. Without careful selection of samples for analysis or data normalization procedures, DNA microarray experiments yield a large number of pseudopositive and pseudonegative results We therefore adopted the strategy of background-matched population (BAMP) screening, in which the sample characteristics are matched as closely as possible, with the exception of the feature of interest (in this case, transformation), before microarray analysis. To achieve this goal, we purified pancreatic carcinoma cells and normal ducta[ cells from pancreatic juice with the use of affinity chromatography based on the shared surface marker MUC1. Analysis of these background-matched samples with DNA microarrays representing 33000 human genes resulted in the identification of several carcinoma-specific genes, including those for AC133 and carcinoembryonic antigen-related cell adhesion molecule 7 (CEACAM7) Cancer-specific expression of these genes was confirmed by quantitative real-time polymerase chain reaction analysis. Microarray analysis with purified pancreatic ducta[ cells has thus provided a basis for the development of a sensitive method for the detection of PDC that relies on pancreatic juice, which is routinely obtained in the clinical setting

6954 Evaluation of cytology and molecular analysis in ercp sampling in the diagosis of pancreatic carcinoma.

In all cases, both conventional cytotlogy and detection of telomerase activity in pancreatic juice obtained at ERCP were performed. Seventeen out of 60 patients was proben at surgery. Final diagnosis was advanced carcinoma (n=5), intraepithelial carcinoma (n=6), chronic pancreatitis(n=4) and endocrine tumor(n=2). Results: Cytology was correctly diagnosed in 4 out of 6 intraepithelial carcinoma. Telomerase activity was detected in 5 out of 6 cases. Using both cytology and telomerase activity, all intraepithelial carcinoma were correctly diagnosed. In all 4 chronic pancreatitis, misinterpretation of conventional cytology was occurred. Conclusion: Molecular analysis of cytological specimen obtained by ERCP is more sensitive in the detection of early pancreatic carcinoma.

Cholecystokinin regulates the invasiveness of human pancreatic cancer cell lines via protein kinase C pathway

We have previously reported that cholecystokinin (CCK) plays an important role in the invasiveness and the production of matrix metalloproteinase-9 (MMP-9) in two human pancreatic cancer cell lines. In this study we investigated the pathway of the invasiveness associated with MMP-9 of those lines regulated by CCK. Two human pancreatic cancer cell lines were treated with CCK-8 alone, CCK-8 and staurosporine, or CCK-8 and indomethacine. The invasiveness and the production of MMP-9 were decreased with staurosporine but not indomethacine. These results suggest that CCK may regulate the invasiveness and the production of MMP-9 via protein kinase C in human pancreatic cancer cell lines.

Telomerase activity for the preoperative diagnosis of pancreatic cancer

Among patients with cancer, those with pancreatic cancer have one of the lowest 5-year survival rates (1). Pancreatic cancer is usually diagnosed by ultrasonography, computed tomography, endoscopic ultrasonography, endoscopic retrograde cholangiopancreatography (ERCP), and cytology (2). However, it is sometimes difficult to distinguish benign from malignant tumors with the use of these diagnostic tools (3,4). K-ras mutations in pancreatic secretions have been reported to be useful for the diagnosis of cancer (5) but were also detected in chronic pancreatitis (6). Therefore, K-ras mutations are not considered to be a specific marker for pancreatic cancer. Telomerase is an enzyme that contains an RNA template complementary to the short DNA sequence repeats (GGTTAG in humans) present at chromosomal ends (i.e., telomeres); the enzyme is believed to be involved in the de novo synthesis at those sites (7). A highly sensitive polymerase chain reaction-based telomerase assay called TRAP (Telomeric Repeat Amplification Protocol) was developed (8,9), and led to the detection of telomerase activity in almost all cancer tissues (10–12). Recently, in surgically resected pancreatic tissue specimens, we detected telomerase activity in 41 (95%) of 43 cancer specimens but none (0 of 11) in benign tumor specimens (13). These findings suggest that telomerase activity in pancreatic duct cells may be useful for cancer diagnosis prior to surgery. Therefore, in this study, telomerase was assayed by use of pancreatic duct cells obtained preoperatively. In almost all cases of suspected pancreatic tumors, ERCP is performed and pancreatic duct cells are collected by use of endoscopic retrograde pancreatic duct brushing (ERPDB). ERPDB has been shown to be a useful method for obtaining cells from the sites of pancreatic duct abnormalities (4). From January 1996 through June 1997, pancreatic duct cells were collected by use of ERPDB from 32 patients with pancreatic duct abnormalities at the Hiroshima University Hospital under informed consent. Institutional guidelines for the use of patients’ materials were followed. The 21 male and 11 female patients ranged in age from 34 to 78 years (mean, 62 years). Part of each specimen was processed for cytologic analysis with the use of Pap staining and was then examined by a pathologist (F. Shimamoto) who made a diagnosis according to guidelines reported by Robins et al. (14). After washing the remaining cells with phosphate-buffered saline, the TRAP assay was performed as described previously (13). In a preliminary study, we examined telomerase activity without making an adjustment for cell number, and weak telomerase activity was detected in three of three chronic pancreatitis cases with massive infiltration of lymphocytes when we used more than 10 cells (data not shown). We re-

Comparative studies of epidermal growth factor, epidermal growth factor receptor, K-ras, and P53 in gallbladder carcinomas with and without an anomalous junction of pancreticobiliary duct

AIM: We reported a 6 to 10 times higher incidence of gallstone disease in curatively gastrectomized patients for cancer (Gastroenterology 104: A362, 1993) and a possible factor for the lithogenesis to be bile infection (Gastroenterology 106: A340, 1994). The aim of this study is to experimentally investigate whether bile infection and postgastrectomy gallstone disease is related with methods of gastrectomy for cancer, i.e., a conservative pylorus-preserving gastrectomy (PPG) and a radical Billroth II subtotal gastrectomy (B-II). METHOD: Five mongrel dogs underwent PPG with the truncal vagi and the hepatic branch intact and 6 dogs underwent B-I1 with wide bursectomy and bilateral truncal vagotomy. Other 6 dogs undergoing only laparotomy (sham operation) were served as the controls. The dogs were maintained with an ordinary laboratory canine diet for 1 year. Gallbladder (GB) bile was collected by sterile fine needle puncture during laparotomy at the time of surgery, 6 months and 12 months. The obtained bile was cultured for bacteriology and analyzed for chemical constituents. Bile acids were quantified by GLC. The presence or absence of gallstone (GS) was consecutively examined by ultrasound at 2 week intervals and by inspection of the aspirated bile. GSs were examined with infrared spectral analysis for classification. RESULTS: The PPG and control dogs incurred no bile infection nor gallstone disease. Three Of the 6 B-II dogs incurred bile infection (E. coli, Bacteroides and Enterococcus). GSs (black stones containing calcium bilirubinate) developed in 3 of the 6 B-II dogs (1 identified at 6 months and 2 at 12 months). Two of the 3 B-II dogs with GS were with bile infection but one without. Total bile acids and bile acid fractions did not significantly differ among the three animal groups. Free bile acids was detected in all of the B-II dogs and in 2 of the 5 PPG dogs but in none of the control dogs. Lithogenicity calculated was within the micellar zone for all dogs. CONCLUSION: Calcium bilirubinate stone developed only in the B-II gastrectomized dogs. Positive bacterial culture and detection of free bile acids were most common in the B-II dogs, which suggested that (1) bile infection is involved in postgastrectomy gallstone disease, (2) B-II subtotal gastrectomy is more liable to bile infection and gallstone disease and (3) PPG may preserve a better sphincter of Oddi motor function, thereby preventing retrograde biliary infection. COMPARATIVE STUDIES OF EPIDERMAL GROWTH FACTOR, EPIDERMAL GROWTH FACTOR RECEPTOR, K-RAS, AND P53 IN GALLBLADDER CARCINOMAS WITH AND WITHOUT AN ANOMALOUS JUNCTION OF PANCREATICOBILIARY DUCT. Keiji Hanada, Masaki Itoh, Kiyomu Fujii, Akira Tsuchida, Shouhei Ishimaru, Manabu Hirata, Toshiyasu Iwao, and Goro Kajiyama. First Dept. of Internal Medicine, Hiroshima University School of Medicine, Hiroshima, Japan. AIMS. Previously, we reported that mutations of Kras and p53 contributed to carcinogenesis in gallbladder mucosa with an anomalous junction of the pancreaticobiliary duct (AJPBD). In this study, we examined the expression of epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR), and mutations of K-ras or p53in gallbladder carcinoma with and without AJPBD. METHODS. We used l0 gallbladder carcinomas with AJPBD (Group A), and 25 gallbladder carcinomas without AJPBD (Group B). EGF and EGFR were examined using immnohistochemistry. Genomic DNA was extracted from routinely processed tissues. Polymerase chain reaction (PCR) restriction fragment length polymorphism was performed for mutations of K-ras. Mutations of p53 were examined using PCR single strand conformation polymorphism. RESULTS. The percentage of immunoreactivity for EGF and EGFR in Group A (70%) was higher than that in Group B (40%). The incidence of mutation of Kras in Group A (30%) was higher than that in Group B (7%). As for p53, although mutations were found in only exon 7 and 8 in Group A, they were found in exon 5, 6, and 7 in Group B. CONCLUSIONS. i) The autocrine loop of EGF and EGFR may play an important role in tumor growth of gallbladder carcinomas associated with AJPBD. 2) Mutations of K-ras may strongly contribute to carcinogenesis of gallbladder carcinomas associated with AJPBD. 3) Mutation spots of p53 in gallbladder carcinomas associated with AJPBD may be different from that in gallbladder carcinomas without AJPBD.