Noriyuki Uesugi

Microsatellite instability and mutation of mitochondrial and nuclear DNA in gastric carcinoma

BACKGROUND & AIMS Microsatellite instability (MSI) in mitochondrial DNA (mtDNA) is observed in some colorectal carcinomas. We attempted to determine if mitochondrial MSI (mtMSI) and mutations occur in gastric carcinomas and if the mtMSI phenotype underlies specific clinicopathologic profiles. METHODS Sixty-two gastric carcinomas (34 intestinal and 28 diffuse types) were investigated. Coding mutations in 8 different mitochondrial genes, mtMSI in a noncoding (C)n tract, and p53 gene mutations were examined by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis. MSI in nuclear DNA (nMSI) and loss of the p53 gene were examined using microsatellite markers. RESULTS Ten of 62 (16%) carcinomas showed the mtMSI phenotype. Mitochondrial gene mutation was detected in 5 carcinomas, 4 of which also showed the mtMSI phenotype. There was a positive correlation between mtMSI and nMSI status. In intestinal carcinomas, mtMSI, nMSI, and p53 gene alterations were frequently detected from early to advanced stages. In diffuse carcinomas, both kinds of MSI were found in only advanced (subserosal or serosal invasion) carcinomas. Six of 7 carcinomas with the nMSI phenotype and all 5 carcinomas with mitochondrial coding mutations had a considerable intestinal-type tumor cell component. CONCLUSIONS Mitochondrial gene mutations, which are associated with the mtMSI phenotype, may play a specific role in the tumorigenesis of intestinal-type gastric carcinomas.

Significance of Ductal Margin Status in Patients Undergoing Surgical Resection for Extrahepatic Cholangiocarcinoma

ObjectivesThe objective of this study was to determine whether carcinoma in situ at the bile duct margin is prognostically different from residual invasive carcinoma in patients with extrahepatic cholangiocarcinoma.Summary Background DataAlthough there are many reports that the ductal margin status at bile duct resection stumps is a prognostic indicator in patients with extrahepatic cholangiocarcinoma, some patients who undergo resection with microscopic tumor involvement of the bile duct margin survive longer than expected.MethodsA retrospective clinicopathological analysis of 128 patients who had undergone surgical resection for extrahepatic cholangiocarcinoma was conducted. The status of the bile duct resection margin was classifiedas negative in 105 patients (82.0%), positive for carcinoma in situ in 12 patients (9.4%), and positive for invasive carcinoma in 11 patients (8.6%).ResultsDuctal margin status was an independent prognostic indicator by both univariate (p = 0.0022) and multivariate (p = 0.0105) analyses, along with lymph node metastasis. There was no significant difference between patients with a negative ductal margin and those with a positive ductal margin with carcinoma in situ (p = 0.5247). The 5-year survival rate of patients with a positive ductal margin with carcinoma in situ (22.2%) was significantly better (p = 0.0241) than with invasive carcinoma (0%). There was a significant relationship between local recurrence and ductal margin status (p = 0.0401).ConclusionsAmong patients undergoing surgical resection for extrahepatic cholangiocarcinoma, invasive carcinoma at the ductal resection margins appears to have a significant relation to local recurrence and also a significant negative impact on survival, whereas residual carcinoma in situ does not. Discrimination whether carcinoma in situ or invasive carcinoma is present is important in clinical setting in which the resection margin at the ductal stump is positive.

Genetic Changes in Primary Colorectal Cancer by Comparative Genomic Hybridization

Comparative genomic hybridization (CGH) is a powerful new technique for the molecular cytogenetic analysis of cancer. In this method, at first the cancer DNA and normal DNA are labeled with biotin and digoxigenin, respectively, and then the labeled DNAs are applied onto normal lymphocyte metaphase preparations in hybridization. After hybridization, they are stained with FITC and rhodamine, respectively, so chromosomal gains and losses in cancer are thus detected by using a green:red ratio. In this study, we analyzed the abnormal chromosomes in nine cases with human primary colon cancer. A gain in chromosomes 11p, 12q, 16p, 20p, and 20q were observed, while a loss of 18q and 22q were discovered. CGH may thus provide us with important information for analyzing the genes in colon cancer.

Three independent genetic profiles based on mucin expression in early differentiated-type gastric cancers—a new concept of genetic carcinogenesis of early differentiated-type adenocarcinomas

Recent molecular studies have shown that the genetic profiles of differentiated-type adenocarcinomas of the stomach are associated with distinct cellular mucin phenotypes (gastric- intestinal- and mixed-phenotypes). Therefore, we examined whether these cellular mucin phenotypes reflect specific molecular genetic alterations, and whether the phenotypes can be used to help categorize the intramucosal neoplasias of gastric tumors. We subclassified tumors into four cellular phenotypes using immunohistochemical mucin analysis. In all, 62 early gastric carcinomas (gastric-phenotype, 13; intestinal-phenotype, 17; mixed-phenotype, 31; unclassified-phenotype, 1) were examined using a combination of polymerase chain reaction microsatellite assays and immunohistochemical analysis in order to detect chromosomal allelic losses of multiple cancer-related chromosomal loci (1p, 3p. 4p, 5q, 8p, 9p, 13p, 17p, 18q and 22q), microsatellite instability (MSI), and overexpression of the p53 protein. In addition, we analyzed the relationship between MSI status and hMLH1 promoter hypermethylation, which is thought to be a cause of high MSI status. For gastric phenotype cancers, the frequency of 3p allelic loss was higher than that of other microsatellite markers, whereas 5q allelic loss was frequently found in intestinal phenotype cancers. The genetic profile of mixed phenotype cancers is comprised of two distinct genetic types: LOH and MSI types. In the former, 5q, 3p and 18q allelic losses are seen frequently in intramucosal carcinomas. On the other hand, 17p, 1p and 9p allelic losses are associated with the development of submucosal carcinomas. MSI was observed only in mixed phenotype cancers (six of 31 mixed phenotype cancers). Overexpression of the p53 protein is common in differentiated-type gastric cancers. In addition, the MSI status of the tumor cells was correlated with the extent of hypermethylation of the hMLH1 promoter. We suggest that the cellular mucin phenotypes of the differentiated-type adenocarcinomas result from distinct genetic alterations.

Microsatellite instability in gallbladder carcinoma: two independent genetic pathways of gallbladder carcinogenesis

Abstract: Although the genetic basis for gallbladder carcinogenesis has not been clarified, considerable evidence has shown that genetic alterations play an important role in the development and progression of human cancers. In this study, we analyzed 30 gallbladder carcinomas to investigate the role of genetic alterations in their tumorigenesis, and to study correlations with their clinicopathological features. Tissue samples were obtained from 30 patients with gallbladder carcinoma (11 men and 19 women; mean age, 62 years; age range, 38–80 years). Genomic DNAs were extracted from fresh tumor tissue. We examined loss of heterozygosity (LOH) in the p53, APC, DCC, RB, and NM23-H1 gene regions by polymerase chain reaction (PCR)-LOH assay using an automated fluorescent DNA sequencer employing four microsatellite markers (p53, APC, DCC, NM23-H1). Five additional microsatellite markers were used for the determination of microsatellite instability (MSI). LOH was found at p53 in 9 of 15 informative cases (60%), at DCC in 10 of 22 (45%), at APC in 5 of 15 (33%), at RB in 1 of 8 (13%), and at NM23-H1 in 1 of 15 (7%). MSI was observed in 5 of 30 cases (17%) in at least one chromosomal loci of these nine microsatellite markers. None of the patients with MSI-positive tumors showed lymph node metastasis, and there was an inverse correlation between MSI and the presence of LOH in gallbladder carcinoma. These results suggest that there are two independent genetic pathways in gallbladder carcinogenesis; that is, an MSI pathway and an LOH pathway.

Aberrations of the K-ras, p53, and APC genes in extrahepatic bile duct cancer.

The genetic alterations involved in extrahepatic bile duct (EHBD) cancer are poorly understood. Our aim was to identify aberrations of the K‐ras, p53, and APC genes in EHBD cancer.

Genetic alterations in DNA diploid, aneuploid and multiploid colorectal carcinomas identified by the crypt isolation technique

Loss of heterozygosity (LOH) and microsatellite instability (MSI) commonly occur in colorectal carcinomas. However, the role of these genetic alterations in determining DNA ploidy status of tumors (diploid, aneuploid and multiploid) remains unclear. In the present study, we attempted to clarify the relationship between genetic alterations and DNA ploidy status. Crypt isolation coupled with DNA cytometric sorting and polymerase chain reaction assay (17 microsatellite markers) were used to study allelic losses and MSI in 59 colorectal carcinomas (diploid, 15; aneuploid, 10 and multiploid, 34). Of the 15 diploid carcinomas, 6 exhibited MSI in which allelic losses were rarely found. The other 9 diploid tumors mostly exhibited allelic losses, but none displayed MSI status. Whereas allelic losses frequently occurred in the aneuploid carcinomas and the aneuploid populations of multiploid carcinomas, they were rarely detected in the diploid populations of multiploid carcinomas. MSI status was not observed in aneuploid carcinomas nor in either population of multiploid carcinomas. Although multiploid carcinomas genetically resemble aneuploid carcinomas in the expression of the severe LOH phenotype, the genetic alterations seen in the diploid populations of multiploid carcinomas may differ from those of diploid carcinomas. Furthermore, all diploid, aneuploid and both the diploid and aneuploid fractions of the multiploid tumors that were non‐MSI exhibited a high rate of LOH, suggesting that LOH is independent of the tumors ploidy status. Int. J. Cancer 88:614–619, 2000.

Usefulness of endoscopic treatment for duodenal adenoma.

In recent years, due to the increasing prevalence of upper gastrointestinal endoscopy, there have been an increasing number of reports on duodenal adenoma and early stage cancer. However, endoscopic techniques for the resection of duodenal adenomas are difficult, due to the anatomical features of the duodenum, and the long distance to the lesion. There have only been a few reports on the use of endoscopic techniques for duodenal adenomas compared to those focused on the stomach and large intestine. For duodenal adenomas, we used a conventional endoscope for lesions proximal to the major duodenal papilla, and a short‐type double balloon endoscope for lesions distal to the papilla. The en‐bloc resection rate was 93.8%. There was only one case of microperforation. Endoscopic manipulation is considered difficult in the deep areas of the duodenum, but double balloon endoscopy enabled stable manipulation and successful resection of the tumor in the majority of cases.

Overexpression of histone deacetylase 6 contributes to accelerated migration and invasion activity of hepatocellular carcinoma cells.

Histone deacetylase 6 (HDAC6) is a cytoplasmic enzyme that regulates many important biological processes, including cell migration, viral infection and autophagy. The aim of this study was to investigate the significance of HDAC6 in the invasion and metastasis activities of hepatocellular carcinoma (HCC). Three HCC cell lines and two primary cultures of hepatocytes were used for biological experiments. Immunohistochemistry for HDAC6 protein was also examined in 70 resected primary HCCs. Knockdown of the HDAC6 gene in the HCC cell lines was carried out by treatment with siRNA, and their migration and invasion activities were examined by the scratch assay and Matrigel invasion assay, respectively. HDAC6 expression was greater in all of the HCC cell lines compared to the primary cultures of hepatocytes. Knockdown of HDAC6 markedly downregulated the migration and invasion activities of all HCC cell lines (P<0.05). Overexpression of HDAC6 protein to a level higher than that in the corresponding normal hepatocytes was observed in 14 (20%) of the 70 primary HCCs, and was significantly correlated with high clinical stage, number of tumors, vascular invasion and intrahepatic metastasis (P<0.05). These results suggest that overexpression of the HDAC6 protein is involved in the migration and invasion activities of HCC cells, and may be a good biomarker for prediction of intrahepatic metastasis.

Experimental Evaluation of Bursting Pressure in Lymphatic Vessels with Ultrasonically Activated Shears

To evaluate the ability of ultrasonically activated shears (USAS) to occlude lymphatic vessels, we investigated the bursting pressure of lymphatic vessels occluded with USAS and compared with pressures in an artery and a vein. The inguinal lymphatic vessels, testicular arteries, and testicular veins were removed from male pigs. The vessels were occluded by USAS at power level 2. The bursting pressures of the harvested vessels were measured ex vivo. The bursting pressure of the artery (average, 1154 mmHg; range, 1000-1341 mmHg) was significantly greater than that in the vein (average, 747 mmHg; range, 560-973 mmHg; p = 0.0024) or the lymphatic vessel (average, 610 mmHg; range, 491-996 mmHg; p = 0.0003). There was no significant difference of bursting pressure between the vein and lymphatic vessel (p = 0.2226). Although the bursting pressures of the vein and lymphatic vessel were significantly lower than that of the artery, they were much higher than that found in physiologic conditions. USAS is considered to be a powerful tool for lymph node dissection for malignant diseases in open surgery as well as laparoscopic surgery.