Takayuki Suto

Promoter hypermethylation of DAP‐kinase is associated with poor survival in primary biliary tract carcinoma patients

To clarify the clinicopathological significance of promoter hypermethylation of tumor suppressor and tumor‐related genes in biliary tract carcinomas, we examined the promoter methylation status of multiple genes in primary biliary tract carcinomas. These consisted of carcinomas of the bile duct, gallbladder, and duodenal ampulla. Surgical specimens were obtained from a total of 37 patients with biliary tract carcinoma. The cohort consisted of 23 patients with bile duct carcinoma, 9 patients with gallbladder carcinoma, and 5 patients with ampullary carcinoma. The methylation status of CHFR, DAP‐kinase, E‐cadherin, hMLH1, p16, RASSF1A, and RUNX3 was examined by methylation‐specif ic polymerase chain reaction (MSP). The correlation between methylation status and clinicopathological characteristics was then assessed. The methylation frequencies of CHFR, DAP‐kinase, E‐cadherin, hMLH1, p16, RASSF1A, and RUNX3 genes were 16.2%, 21.4%, 27.0%, 8.1%, 24.3%, 27.0%, and 56.8%, respectively, in primary biliary tract carcinomas. The number of methylated genes per sample was 2.17±0.28 (average±SD) in bile duct carcinomas, 1.80±0.97 in ampullary carcinomas, and 0.89±0.35 in gallbladder carcinomas, with a statistically significant difference between bile duct carcinomas and gallbladder carcinomas (P=0.02). As for clinicopathological significance, patients with a methylated RUNX3 promoter were significantly older than those with unmethylated RUNX3 (P=0.01), and DAP‐kinase methylation was more frequent in poorly differentiated tumors than in well to moderately differentiated ones (P=0.04). The overall survival rate was significantly lower in patients with methylated DAP‐kinase (P=0.009) or RUNX3 (P=0.034) compared to those with unmethylated genes. Furthermore, DAP‐kinase methylation‐positive status was independently associated with poor survival in multivariate analyses (hazard ratio=8.71, P=0.024). A significant proportion of primary biliary tract carcinomas exhibited promoter hypermethylation of tumor suppressor and tumor‐related genes, although bile duct carcinomas are more prone to being affected by promoter methylation than are gallbladder carcinomas. Hypermethylation of DAP‐kinase appears to be a significant prognostic factor in primary biliary tract carcinomas.

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.

Infrequent microsatellite instability in biliary tract cancer

Microsatellite instability (MSI) has been reported in several tumors. However, few reports are available concerning MSI in biliary tract cancers. We investigated MSI and allelic loss at the hMLH1 and hMSH2 gene loci in biliary tract cancers.

ROLE OF DNA ANEUPLOIDY, OVEREXPRESSION OF P53 GENE PRODUCT, AND CELLULAR PROLIFERATION IN THE PROGRESSION OF GASTRIC CANCER

DNA aneuploidy, p53 overexpression, and high cell proliferation frequently occur in gastric cancer. However, little is known about the time of their appearance throughout cancer progression. Therefore, the objective of the present study was to determine when such abnormalities occur during gastric cancer progression. We classified the gastric cancers examined into intestinal (n = 65) and diffuse (n = 34) types. DNA ploidy was examined using flow cytometry and expression of MIB-1 and p53 immunoreactivity were studied using the avidin-biotin complex method in three stages of gastric cancer (mucosal, submucosal, deeply invasive cancer, i.e., advanced cancer). The incidence of DNA aneuploidy in intestinal-type mucosal cancers (15/27, 55.6%) was lower than that of submucosal invasive cancers (14/16, 87.5%) or advanced cancers (19/22, 86.4%), while a low incidence of DNA aneuploidy was observed in each diffuse-type cancer group (mucosal, 1/12, 8.3%; submucosal invasive, 3/9, 33.3%; advanced, 8/14, 57.1%). Although overexpression of the p53 gene in intestinal-type cancer was found in early stage, that in diffuse-type cancer was observed in advanced stage. Among the intestinal-type mucosal cancers, the MIB-1 percent positive was higher in aneuploid tumors than diploid ones. DNA aneuploidy and overexpression of the p53 gene may play an important role in the early tumorigenesis of intestinal-type gastric cancer and in the late event of tumorigenesis of diffuse-type gastric cancer.

Laparoscopic Excision of Retroperitoneal Tumors: Report of Three Cases

Retroperitoneal neural tumors are rarely excised laparoscopically, with fewer than ten cases reported in the literature. Between February 2005 and December 2007, we performed successful planned laparoscopic excision of retroperitoneal tumors using the four-trocar technique in three patients. All three patients were women, with a mean age of 40.7 years. The mean tumor size was 4.8 cm. The mean operative time was 126 min and the mean blood loss 14.3 ml. The postoperative pathological diagnosis was schwannoma in one patient and ganglioneuromas in two. There was no morbidity or mortality. Although difficult to diagnose preoperatively, neural tumors in the retroperitoneal space are most often benign, with a good prognosis. Laparoscopic surgical techniques for retroperitoneal tumors are safe, and their use is encouraged when an appropriate diagnosis is made, after exclusion of malignant subtypes.

Correlation of histologic morphology and tumor stage with molecular genetic analysis using microdissection in gastric carcinomas.

Precise correlation of histomorphology with the results of molecular genetic analysis is difficult in gastric cancer tissue composed of intestinal and diffuse types. A novel microdissection procedure was applied to correlate p53 and APC allelic loss with histologic type and tumor stage (mucosal vs. invasive cancer) in formalin-fixed, paraffin-embedded specimens of 25 gastric cancers. In addition, mucosal and invasive lesions were dissected from each of 11 invasive gastric cancers to study progression, and allelic loss of the p53 and APC genes was assessed. The p53 gene underwent loss of heterozygosity (LOH) in 4 of 4 informative cases of intestinal-type gastric cancer with mucosal lesions associated with invasion. By contrast, no p53 LOH was found among 6 informative cases with mucosal cancer. LOH of the APC gene in both intestinal and diffuse types of cancer was detected in 4 of 7 and 5 of 6 informative cases, respectively. These data suggest that allelic deletion of the p53 gene in intestinal-type gastric carcinoma predicts the invasive potential of mucosal cancer, and that in-activation of the APC gene plays a role in the genetic tumorigenesis of both intestinal and diffuse types of gastric cancer. Microdissection can correlate genetic alterations with histologic morphology in gastric cancer.

Immunohistochemical detection of proliferating cell nuclear antigen and p53 expression in carcinoma of the extrahepatic bile duct.

We used an immunohistochemical method to determine the concentrations of p53 and proliferating cell nuclear antigen (PCNA) to evaluate their usefulness as a predictor of malignancy and examined the relationship between PCNA and p53 in carcinomas of the extrahepatic bile duct (EHBD). Paraffin-embedded specimens from 46 patients were immunostained for PCNA and p53 using PC10 and DO7 monoclonal antibodies, respectively. The PCNA labeling index (LI) was closely associated with the stages of the tumor and depth of invasion (p < 0.05). The cumulative survival rate of patients with a low PCNA LI (LI < 47%) was found to be significantly better than that of patients with a high PCNA LI (LI > or = 47%) in all cases and patients with advanced cancer by univariate analysis (p < 0.05), but PCNA LI was not an independent prognostic factor in multivariate analysis. We detected p53 in 37% of the EHBD cancers. We also found that p53 positivity was not related to the percentage of PCNA-labelled cells or survival. The results suggest that PCNA immunoreactivity may be a useful predictor of malignancy in patients with EHBD carcinomas.

DNA mapping of gastric cancers using flow cytometric analysis

Although numerous studies of gastric cancers on DNA ploidy have been reported, differences in the degree of aneuploidy (DNA index, DI) during progression have not been identified. We attempted to chart the differences in DIs during progression to clarify the role of aneuploidy in gastric cancers. We classified the gastric cancers examined into intestinal (n = 88) and diffuse (n = 48) types, and then analyzed 136 gastric cancers (intramucosal cancer, 42; submucosal cancer, 39; advanced cancer, 55) by flow cytometry using multiple sampling. In addition, we examined the DNA ploidy pattern of mucosal and submucosal lesions using the same submucosal cancers to study the tumor progression in individual cancers. Intratumoral DNA differences in DNA ploidy were observed in both types of gastric cancers. In intestinal-type cancers, multiple subclones indicated by a different DI occurred during the early stage of gastric cancers, whereas in diffuse-type cancers, multiple subclones were found primarily in advanced cancers. Although the DI varied widely in early intestinal-type cancers between 1.0 and 2.0, in early diffuse-type cancers, the DI tended to be less than 1.2. However, in advanced stage gastric cancers, the DI distribution was similar for both histological types. In intestinal-type cancers, high DI (>1.3) aneuploidy was frequently found in mucosal lesions. In contrast, only low DI (<1.2) aneuploid clones were observed in mucosal lesions of diffuse-type cancers. The present results suggest that high DI aneuploid tumor clones in intramucosal cancers acquire invasive ability when they progress to submucosal cancers, whereas DNA aneuploidy itself plays an important role in submucosal invasion of diffuse-type cancers.

Pure Laparoscopic Left Hemihepatectomy for Hepatic Peribiliary Cysts with Biliary Intraepithelial Neoplasia.

Introduction. Hepatic peribiliary cysts (HPCs) usually originate due to the cystic dilatation of the intrahepatic extramural peribiliary glands. We describe our rare experience of pure laparoscopic left hemihepatectomy (PLLH) in a patient with HPCs accompanied by a component of biliary intraepithelial neoplasia (BilIN). Case Presentation. A 65-year-old man was referred for further investigation of mild hepatic dysfunction. Contrast-enhanced computed tomography showed dilatation of the left-sided intrahepatic bile duct, and biliary cytology showed class III cells. The patient was highly suspected of having left side-dominated cholangiocarcinoma and underwent PLLH. Microscopic findings revealed multiple cystic dilatations of the extramural peribiliary glands; hence, this lesion was diagnosed as HPCs. The resected intrahepatic bile duct showed that the normal ductal lumen comprised low columnar epithelia; however, front formation on the BilIN was observed in some parts of the intrahepatic bile duct, indicating that the BilIN coexisted with HPCs. Conclusion. We chose surgical therapy for this patient owing to the presence of some features of biliary malignancy. We employed noble PLLH as a minimally invasive procedure for this patient.

Laparoscopic umbilical hernia repair in a cirrhotic patient with a peritoneovenous shunt

A 62‐year‐old Japanese woman who had developed massive cirrhotic ascites was referred to our hospital for a peritoneovenous shunt implant. However, CT examination revealed an umbilical hernia that had not been observed before the peritoneovenous shunt was implanted. We decided to perform laparoscopic umbilical hernia repair to keep carbon dioxide from flowing backward into the central circulatory system. We first clamped the catheter and set the upper limit of the pneumoperitoneum pressure to 6 mmHg. The central venous pressure was also measured simultaneously. Mesh was then applied over the hernia and fixed by the double‐crown technique. Finally, 1000‐mL physiological saline was infused into the abdominal cavity while the pneumoperitoneum was slowly released. In this case, we safely performed laparoscopic umbilical hernia repair while making some alterations, specifically catheter clamping, reducing pneumoperitoneum pressure, monitoring central venous pressure, and infusing physiological saline.