Kiyomu Fujii

K-ras and p53 mutations in stage I gallbladder carcinoma with an anomalous junction of the pancreaticobiliary duct.

An anomalous junction of the pancreaticobiliary duct (AJPBD) was thought to be an important risk factor for gallbladder carcinoma in Japan. In this report, we compared K‐ras and p53 mutations in Stage I gallbladder carcinomas (GC) of patients with AJPBD with those in patients without AJPBD.

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.

Reg IV expression is associated with cell growth and prognosis of adenoid cystic carcinoma in the salivary gland.

Aims:  Regenerating islet‐derived family, member 4 (Reg IV) is associated with the progression of various cancers. The aim was to examine Reg IV expression in adenoid cystic carcinomas (ACCs) in salivary glands.

Immunohistochemical analysis of nm23 gene product in human gallbladder carcinomas

The expression of nm23, the product of a candidate suppressor gene for tumour metastasis, was examined immunohistochemically in human gallbladder carcinomas and compared with clinicopathological features. Seventy-eight (72%) of 107 carcinomas expressed nm23 protein regardless of histological type, while non-neoplastic mucosa occasionally showed very weak immunoreactivity to nm23. No obvious correlation was observed between nm23 protein expression and depth of tumour invasion or tumour stage. The expression of nm23 protein was detected in 60% and 74% of the cases with and without lymph node metastasis, respectively, indicating no relationship to metastatic ability. Fifty-eight percent of the cases showed reduction of nm23 immunoreactivity in tumour cells invading the stroma at the border of tumour cell nests compared with cells at the centre of the tumour. Only 7% of the cases showed increased nm23 expression in tumour cells at the border. These results suggest that in gallbladder carcinoma decreased expression of nm23 may not have implications for metastasis but may play a part in local invasion.

EXPRESSION OF CRIPTO IN HUMAN GALL BLADDER LESIONS

The expression of cripto, a member of the epidermal growth factor (EGF) family, was examined by immunohistochemistry in benign lesions and carcinomas of the gall bladder. Cripto expression was detected in 6 (67 per cent) of 9 hyperplasias, 4 (58 per cent) of 7 adenomas, and 89 (68 per cent) of 132 adenocarcinomas of the gall bladder. The degree of cripto expression was not correlated with depth of tumour invasion, tumour stage or patient prognosis. The incidence of cases with cripto expression was significantly higher in papillary and well‐differentiated adenocarcinomas (positive 73 per cent; strongly positive 38 per cent) than in moderately and poorly differentiated adenocarcinomas (positive 54 per cent; strongly positive 17 per cent) (P<0·05). These results suggest that cripto expression may not relate to progression in gall bladder carcinomas, but may be associated with tumour differentiation.

Expression of the proliferative cell nuclear antigen (PCNA) in adenocarcinoma of the gallbladder, and its relationship to prognosis

Proliferative cell nuclear antigen (PCNA) has been correlated with degree of differentiation in some tumours, but information on PCNA expression in adenocarcinoma of the gallbladder is currently limited. Therefore, we examined PCNA expression in adenocarcinoma of the gallbladder, and its relationship to prognosis. The expression of PCNA was studied by immunohistochemistry in 70 formalin-fixed, paraffin-embedded specimens of surgically removed adenocarcinomas of the gallbladder. The percentage of stained nuclei was recorded, and the PCNA-labelling index (LI) was expressed as the ratio of labelled nuclei to the total number of nuclei counted. In all histological types, the PCNA-LI in the invasive zone of the tumour was higher than that in the luminal zone of the tumour (p < 0.05). The PCNA-LI showed a stepwise increase with decreasing degrees of differentiation in both the invasive and the luminal zone of the tumour (p < 0.01). In advanced adenocarcinomas, patients whose tumours had a PCNA-LI of less than 35 in the invasive zone had significantly longer survival rates than those with PCNA-LI equal to or greater than 35 (p < 0.01). Multivariate analysis, using the Cox proportional hazards model, indicated that a PCNA-LI > or = 35 in the invasive zone of the tumour was a significantly unfavourable prognostic factor (p = 0.002). The PCNA-LI of routinely processed specimens of adenocarcinoma of the gallbladder may be helpful for the evaluation of cell proliferation and prognosis.

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.