Kei Shiraishi

A comparison of DNA copy number changes detected by comparative genomic hybridization in malignancies of the liver, biliary tract and pancreas.

Tumors arising from the liver, biliary tract and pancreas, which originate in the foregut and are in close anatomical proximity to each other, sometimes show similar histological features. No studies have focused on genetic similarities and differences between tumors of these organs. To elucidate the similarities and differences in DNA copy number alterations between tumors of these organs, we applied comparative genomic hybridization (CGH) to cancers of the liver (31 cases), biliary tract (42 cases) and pancreas (27 cases). Some alterations were common to tumors of all three organs, and some were preferential in certain types of tumor. Gains of 1q and 8q and losses of 8p and 17p were common to all tumors. In contrast, 13q14 and 16q losses were detected exclusively in hepatocellular carcinomas (HCCs; p < 0.01). The incidence of 17q21 gain and 5q loss was higher in biliary tract cancers than in the other two types (p < 0.05). Pancreatic cancers exhibited higher incidence of 5q14-q23 gain and 19p loss than tumors of other organs (p < 0.01). Gains of 7p, 7q, 12p and 20q and losses of 3p, 6q, 9p and 18q were frequent in both biliary tract and pancreatic cancers but rare in HCCs (p < 0.05). The present results suggest that although genes located at 1q, 8p, 8q and 17p are frequently involved in HCC, biliary tract and pancreatic cancer, at least some of the genes implicated in carcinogenesis are different between these three types. It is also suggested that CGH analysis is useful as a potential adjunct for the diagnosis and management of these tumors of organs that are anatomically close to one another.

Detection of K-ras and p53 gene mutations in pancreatic juice for the diagnosis of intraductal papillary mucinous tumors.

The aim of this study was to investigate mutations of the K-ras oncogene and the p53 tumor suppressor gene in pancreatic juice and to evaluate our method for the diagnosis of intraductal papillary mucinous tumors (IPMT). Pancreatic juice was collected endoscopically from 12 patients with IPMT who underwent surgical resection (eight carcinomas and four adenomas) and eight cases without evident pancreatic diseases. DNA was extracted and both genes were examined by polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis and direct sequencing. In addition, surgically resected specimens were analyzed for both genes by the same methods, and p53 overexpression was investigated immunohistochemically. K-ras point mutations were detected in pancreatic juice from all 12 patients (100%) and p53 mutations were detected in five of 12 (42%). They were detected not only in carcinoma but also in adenoma and there was no difference between the mutations detected in pancreatic juice and surgical specimens. No mutations were found in any cases without pancreatic diseases. These findings suggest that alterations of K-ras and p53 gene are common events in the development of IPMT and that genetic analysis of them in pancreatic juice can be a useful tool for the clinical diagnosis of IPMT before surgery.

Genetic Aberrations Detected by Comparative Genomic Hybridization in Biliary Tract Cancers

In order to elucidate cytogenetic changes characteristic of biliary tract cancer, we examined the genetic imbalances in 18 biliary tract cancers using comparative genomic hybridization (CGH). The most common sites of increases in copy number, in order of frequency, were 17q (33% of the cases), 5p (28%), 3q (22%), 7p (22%), 8q (22%), and 12p (22%), whereas copy number decreases of 6q (28%), 18q (28%), 4q (22%), 5q (22%), and 9p (22%) were frequent. The average number of chromosomal aberrations was significantly greater in stage IV than in stage III tumors (7.9 vs. 2.2/tumor, p < 0.05). The frequent aberrations detected in this study may be related to the development and/or progression of biliary tract cancers. This is the first report on CGH of biliary tract cancers.

Detection of genetic alterations in pancreatic cancers by comparative genomic hybridization coupled with tissue microdissection and degenerate oligonucleotide primed polymerase chain reaction

The aim of this study was to elucidate cytogenetic changes in pancreatic cancers (PCs) and to examine their clinical implications. We screened for genetic alterations in 32 primary PCs including 4 cases with distant organ metastasis using comparative genomic hybridization coupled with tissue microdissection and degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR). The present study revealed frequent gains of chromosomes 13q and 15q and a loss of Xq in addition to a high prevalence of chromosomal imbalances. The average number of total genetic alterations and gains tended to be higher in N1 tumors (TNM classification) than in N0 tumors. The average number of amplifications was significantly higher in M1 tumors than in M0 tumors (p = 0.024). Gain/amplification of 20q was more frequently observed in M1 tumors than in M0 tumors (p = 0.016), and this change was also detected in all of 4 distant metastatic lesions. Losses of 6q, 8p, 9p, 17p, and 18q were recurrent in N0 and M0 tumors, and these alterations were also retained in N1 and M1 tumors. These observations suggest that these genetic losses contribute to the development of PCs and that increases in the DNA copy number confer an aggressive character on cancer cells. Especially, gain/amplification of 20q was associated with the potential of distant organ metastasis of tumor cells.

Evaluation of the Reliability of Chromosomal Imbalances Detected by Combined Use of Universal DNA Amplification and Comparative Genomic Hybridization

Comparative genomic hybridization (CGH) analysis of microscopic tumor samples is allowed by universal DNA amplification using degenerate oligonucleotide primed‐PCR (DOP‐PCR). To evaluate the reliablity of DOP‐PCR CGH, we performed DOP‐PCR CGH and standard CGH in parallel using DNAs extracted from 10 malignant tumors of the hepatobiliary tract and pancreas. Similar results were obtained by both methods with a few exceptions, indicating that DOP‐PCR CGH provides cytogenetic information equivalent to that obtained from standard CGH. We also investigated the sensitivity of DOP‐PCR CGH using sequential dilutions of DNA from microdissected tumor cells. DOP‐PCR using 100 to 800 pg of template DNA yielded successful CGH results. However, less than 50 pg of template DNA was not suitable because of the small amount of generated DNA. These findings suggest that DOP‐PCR CGH is applicable for CGH analysis of tiny specimens which are too small for standard CGH. Accordingly, DOP‐PCR CGH analysis may become a useful method in clinical laboratory examination.

p53 Mutations in Two Patients with Intraductal Papillary Adenoma of the Pancreas

There has been no report on p53 gene mutation in benign human pancreatic intraductal tumors. We examined pancreatic juice and tissue specimens from two patients with intraductal papillary adenoma of the pancreas by polymerase chain reaction‐single‐strand conformation polymorphism analysis and direct sequencing and found point mutations of p53 gene resulting in amino acid substitutions in exons 6 and 8. Thus, p53 gene mutation may be an early event in the neoplastic process of some pancreatic intraductal tumors and may play an important role in tumorigenesis.

Calcium concentration and artificial precipitates in human pancreatic juice.

We studied the role of the increase in the calcium concentration in pure pancreatic juice of alcoholic noncalcified chronic pancreatitis. Pure pancreatic juice was obtained endoscopically. The pancreatic juice from patients with chronic pancreatitis was adjusted to pH 7.5; then the calcium concentration was adjusted to 0.4, 2.9, 5.4, or 10.4 mmol/L. Artificial precipitates were produced by incubation of the samples at 37°C for 6 hours. Proteins in the artificial precipitates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the protein patterns were compared with the patterns of natural protein plugs from patients with chronic pancreatitis. The amount of the precipitate increased as the added calcium increased. The protein patterns of SDS-PAGE of the artificial precipitates were similar to those of protein plugs. Albumin, a-amylase, lipase, trypsinogen, and chymotrypsinogen were identified by immunoblotting both in the precipitate and in the protein plug. The increased calcium concentrations in pancreatic juice induced the formation of precipitates whose protein composition was similar to that of protein plugs. An increased calcium concentration in human pancreatic juice may play an important role in the pathogenesis of protein plugs.