Teruhiko Iwase

Clinical and Genetic Analysis of Noncancerous and Cancerous Biliary Epithelium in Patients with Pancreaticobiliary Maljunction

We analyzed clinical features and genetic alterations in the noncancerous and cancerous biliary lesions obtained from pancreaticobiliary maljunction (PBM) patients. Gallbladder (GB) and bile duct (BD) lesions were obtained surgically from 36 patients with PBM, and polymerase chain reaction (PCR) methods were used to examine for mutations of the K-ras gene and the p53 gene and for microsatellite instability (MSI). The 36 cases were clinically classified into two types according to whether extrahepatic bile duct dilatation was present: a congenital choledochal dilatation (CCD) group (n 20) and a noncongenital choledochal dilatation (NCCD) group (n 16). In the NCCD group, all 16 GB specimens exhibited hyperplastic, dysplastic, and cancerous (n 9) lesions, but no pathological lesions were detected in the 12 BD specimens. On the other hand, in the CCD group, pathological examination revealed lesions, including 8 cancerous lesions, in 60% of the 20 GB specimens and lesions, and including 8 cancerous lesions, in 65% of the 20 BD specimens. K-ras mutations and MSI were detected in 33.3% and 0%, respectively, of 9 hyperplastic lesions, 28.6% and 85.7%, respectively, of 7 dysplastic lesions, and 60.0% and 80.0%, respectively, of 25 cancerous lesions (p < 0.05; MSI in hyperplasia vs. dysplasia and cancer). There was no difference of the frequency in K-ras mutations and MSI between the NCCD and CCD groups. By contrast, p53 mutations were detected only in the cancerous GB lesions of both types, the rate being 35.3%. Genetic alterations of K-ras, MSI, and p53 are strongly associated with biliary tract cancer in PBM patients. MSI appears to contribute to carcinogenesis in the biliary tract mucosa of PBM patients, and p53 mutations may be related to the development of GB cancer in the CCD group. Pancreaticobiliary maljunction (PBM) is a rare malformation defined as anomalous union of the common bile duct with the pancreatic duct. Babbitt et al. [1] suggested that PBM is associated with congenital biliary dilatation; however, there have been some cases of PBM without biliary dilatation [2]. PBM has recently been attracting considerable attention as an important risk factor for biliary tract cancer. In Japan, biliary tract tumors have been reported to develop in 20% to 30% of patients with PBM, a much higher percentage than detected at autopsy or during surgery in the absence of PBM (0.26%–1.8%) [3]. Reveille et al. [4] reported that PBM is a major risk factor for biliary tract tumors, with incidences 5to 35-fold higher than in patients without PBM. This has been attributed to biliary tract epithelial cell damage caused by pancreatic juice reflux into the biliary tract, with the mixture of bile and pancreatic juice causing an increase in mutagenicity [5]. PBM is well known to occur in two major types, according to whether the extrahepatic bile ducts are dilated: a congenital choledochal dilatation (CCD) type and a noncongenital choledochal dilatation (NCCD) type, and the site of cancer development in these two types is quite different. PBM with NCCD is associated with a higher incidence of gallbladder (GB) cancer and a lower incidence of bile duct (BD) cancer, while CCD is associated with both GB cancer and BD cancer [2, 3, 6, 7]. It is well established that the development of human cancer is a multistep process, and molecular biological studies have revealed involvement by many oncogenes and tumor-suppressor genes [8]. Mutations of two of them, K-ras and p53, have been widely examined in various precancerous and cancerous lesions [9–15]. Recently, microsatellite instability (MSI) has also come to be considered a class of genetic alterations that causes human cancer [16, 17]. MSI, which represents replication errors (RERs), results from DNA mismatches due to environmental or hereditary factors and leads to genomic instability and accumulation of abnormalities in oncogenes and/or tumor-suppressor genes. MSI has been reported to contribute to the development of a variety of

Immunohistochemical Localization of Hepatocyte Growth Factor Protein in Pancreas Islet A-Cells of Man and Rats

Hepatocyte growth factor (HGF), a potent mitogen for adult rat hepatocytes in primary culture, has previously been shown to be primarily expressed in the nonparenchymal cells of the liver. Using polyclonal antisera against human and rat HGFs we studied the tissue distribution of HGF immunohistochemically and found the most intense staining in the pancreas islet cells in both man (autopsy cases) and the rat. Differential localization of 4 pancreas islet hormones, glucagon, insulin, somatostatin and pancreatic polypeptide, revealed HGF to be preferentially expressed within the glucagon‐positive cells. The results indicate that HGF is primarily produced or stored in A‐cells and may act as a growth factor in a paracrine and an endocrine fashion, like various other hormones.

Demonstration of ras and p53 Gene Mutations in Carcinomas in the Forestomach and Intestine and Soft Tissue Sarcomas Induced by N‐Methyl‐N‐nitrosourea in the Rat

The presence of ras family and p53 gene mutations in rat forestomach, intestine and liver tumors and soft tissue sarcomas induced by Nmethyl‐N‐nitrosourea (MNU) was examined using polymerase chain reaction‐single strand conformation polymorphism (PCR‐SSCP) followed by direct sequencing analysis. In the forestomach squamous cell carcinomas (SCC), Ha‐ros and p53 mutations were detected in 2 (40%) and 4 (80%) of 5 cases, respectively. The figures for Ki‐ras and p53 gene mutations in adenocarcinomas of the large and small intestines were 3 (18.8%) and 5 (31.3%) of 16 cases. Soft tissue sarcomas in different sites were found to have mutations of Ki‐ras in 7 (23.3%)and of p53 in 9 (30%) of 30 cases. One forestomach SCC and 2 soft tissue sarcomas had double p53 mutations in different exons. Single cases of forestomach SCC and intestinal adenocarcinoma had mutations in both Ki‐ras and p53 genes. No mutations were found in counterpart benign tumors or hepatocellular adenomas. The p53 mutation spectrum revealed preferential clustering within exon 8 for the forestomach SCCs, and exons 5 and 8 for the intestinal adenocarcinomas, whereas the distribution was evenly spread through exons 5 to 8 in soft tissue sarcomas. All the detected ras or p53 mutations were G:C to A:T transitions. These results indicate firstly that specific Ki‐ras, Ha‐ras and p53 gene mutations in MNU‐induced lesions are related to particular alkylation sites (G:C to A:T transitions) and secondly, although not essential, Ki‐ras, Ha‐ras or p53 gene mutations may be involved in the progression stage of forestomach, intestine and soft tissue neoplasms induced by MNU.

A mucous histochemical and immunohistochemical study of precancerous and neoplastic lesions in the human pancreas.

SummaryA total of 44 cases of pancreatic lesions, including hyperplasia (six) cases, adenoma (mucinous cystadenomas [eight] and intraductal papillary adenoma [eight]), noninvasive intraductal papillary tumors (five), and invasive ductal carcinomas (17) were investigated possibly to establish a diagnostic marker. We examined the type of mucin secreted and immunoreactivities of antibodies toras-p21 and c-erbB-2 oncogene products. A significant decrease in the amount of mucin was found in invasive lesions, and this was associated with a shift toward production of neutral mucins and especially sialomucins. Hyperplasia and adenoma, in contrast, demonstrated a predominance of neutral mucin. The sulfated mucins found in normal epithelium were only very weakly stained in any of the tumor types. Thirty-three percent of non-invasive intraductal papillary tumors and 88% of invasive ductal adenocarcinomas demonstrated strong binding of theras-p21 antibody. In contrast no obvious differences in expression of c-erbB-2 were evident between the groups. In conclusion, a combined mucin histochemical/immunohistochemical approach may facilitate accurate diagnosis.

Histogenetic Stereological Reconstruction of Rat Basophilic, Clear, and Oncocytic Neoplastic Renal Cell Lesions Using Carbonic Anhydrase Type II-PAS Double-Stained Sections

The histogenesis of 3 types of rat renal cell tumors (basophilic cell, clear cell, and oncocytic) was stereologically analyzed, with particular attention paid to transitions from normal tubules. Early nitrosamine-induced preneoplastic lesions, including dysplastic tubules (altered tubules), epithelial hyperplasias, and small adenomas, were reconstructed using serially sectioned specimens processed for carbonic anhydrase type II (CA) and periodic acid-Schiff (PAS) (CA-PAS) double staining to allow easier distinction of the nephron segments: Proximal tubules had a PAS-positive brush border and were weakly positive for CA in the cytoplasm; distal tubules were PAS negative and weakly positive for CA; collecting ducts were PAS negative and strongly positive for CA. Similarly, cytochrome c oxidase (CytOx) and CytOx-PAS double staining was also applied to confirm the character of oncocytic lesions. All basophilic lesions (7 of 7) showed transition to proximal tubules. Clear cell lesions positive for CA, on the other hand, showed transition to distal tubules in 4 of 9 (44.4%) lesions and to collecting ducts in 4 of 9 (44.4%) lesions, but in only 1 of 9 (11%) to a proximal tubule. All oncocytic lesions (16 of 16), characterized by positivity for both CA and CytOx, showed transition to collecting ducts. The results indicate that the origins of renal cell neoplasia are proximal tubules for the basophilic cell lesions, either proximal or distal tubules for their clear cell counterparts, and collecting ducts for oncocytic lesions.

Enhanced neoplastic lesion development with adenine-induced experimental multicystic nephropathy by adenine — a model system for the analysis of renal tumor generation in long-term hemodialysis patients

To cast light on the high incidence of renal cell tumors (RCT) in long-term hemodialysis patients, the role of background multicystic nephropathy was studied in a rat model. Group 1 animals were initially given N-ethyl-N-hydroryethylnitrosamine (EHEN) then subjected to adenine feeding until killing during weeks 20-27. Groups 2 and 3 received EHEN and adenine, respectively. All rats receiving adenine developed multicystic nephropathy. The incidence of renal cell hyperplasias (RCH) and multiplicities of both RCH and RCT in Group 1 were significantly increased as compared with Group 2, suggesting multicystic nephropathy provides favorable environment for tumor development.

A Case of Pancreatolithiasis Treated by a Combination of Endoscopic Extraction and Extracorporeal Shock Wave Lithotripsy

We report on the case of a 50‐year‐old woman with idiopathic chronic calcifying pancreatitis and diabetes. An endoscopic retrograde pancreatography showed a stone with a diameter of 23 mm and multiple small stones in the head of the pancreas. An endoscopic pancreatic sphincterotomy was performed. However, the stone could not be removed endoscopically. So we performed an extracorporeal shock wave lithotripsy (ESWL) using a Tripter X1. The stone was located in the shock wave focus by fluoroscopy. Under intravenous sedation, the patient received 5 ESWL sessions (a total of 11700 shock waves with an energy of 18kv). ESWL permitted stone disintegration and successful endoscopic extraction of the fragments. Complete clearance in the main pancreatic duct was achieved. No severe complications were observed. After treatment, an improvement in the PFD test was seen. ESWL is an effective method for treatment of endoscopically unextractable pancreatic ductal stones.

A New Peroral Cholecystoscopy Method Utilizing a Shape Memory Alloy Bending Catheter‐Report of Two Cases

For the purpose of improving the ease and reliability of catheter insertion into the cystic duct and gallbladder, we developed a new combination method utilizing an ultrathin fiberscope (miniscope, 0.8 mm), a guidewire (0.25 inches) and a shape memory alloy, bendable, catheter (SMA catheter, 2.6 mm). The main feature of the SMA catheter, which incorporates a micro‐actuator, is that its tip can be bent by remote control as desired. The SMA catheter could be inserted successfully into the cystic duct using a guide‐wire under direct miniscope vision in two patients with gallbladder lesions.