B-RAF mutation and accumulated gene methylation in aberrant crypt foci (ACF), sessile serrated adenoma/polyp (SSA/P) and cancer in SSA/P.

BACKGROUND: Sessile serrated adenomas/polyps (SSA/Ps) are a putative precursor of colon cancer with microsatellite instability (MSI). However, the developmental mechanism of SSA/P remains unknown. We performed genetic analysis and genome-wide DNA methylation analysis in aberrant crypt foci (ACF), SSA/P, and cancer in SSA/P specimens to show a close association between ACF and the SSA/P-cancer sequence. We also evaluated the prevalence and number of ACF in SSA/P patients. METHODS: ACF in the right-side colon were observed in 36 patients with SSA/Ps alone, 2 with cancers in SSA/P, and 20 normal subjects and biopsied under magnifying endoscopy. B-RAF mutation and MSI were analysed by PCR-restriction fragment length polymorphism (RFLP) and PCR-SSCP, respectively, in 15 ACF, 20 SSA/P, and 2 cancer specimens. DNA methylation array analysis of seven ACF, seven SSA/P, and two cancer in SSA/P specimens was performed using the microarray-based integrated analysis of methylation by isochizomers (MIAMI) method. RESULTS: B-RAF mutations were frequently detected in ACF, SSA/P, and cancer in SSA/P tissues. The number of methylated genes increased significantly in the order of ACF

A novel diagnostic method for evaluation of vascular lesions in the digestive tract using infrared fluorescence endoscopy.

BACKGROUND AND STUDY AIMS: We have developed an infrared fluorescence endoscope to evaluate gastrointestinal vascular lesions. Infrared endoscopy (IRE) after intravenous administration of indocyanine green (ICG) is used at present to examine vascular lesions such as esophageal varices. However, no previous study has compared the sensitivity of infrared fluorescence endoscopy (IRFE) with that of IRE. In this study, we compared the usefulness of IRFE and IRE. PATIENTS AND METHODS: For IRFE we used an infrared endoscope equipped with excitation and barrier filters and an intensified charge-coupled device camera. In preliminary experiments, the observable tissue depth was assessed by wrapping increasing numbers of layers of commercially available pork around a syringe containing a uniform concentration of ICG or by changing the concentration of ICG in a syringe covered by a piece of pork of uniform thickness. In the clinical part of the study, ICG was administered intravenously at different concentrations to patients with esophageal varices and the resulting infrared fluorescent images were evaluated. RESULTS: The preliminary experiments revealed that the depth of tissue that could be visualized was significantly greater in IRFE than it was in IRE (11.2 mm in IRFE vs. approximately 3.2 mm in IRE). Clear infrared fluorescence was obtained by IRFE at lower concentrations of ICG than the concentrations required to obtain clear images using IRE. In the clinical part of the study, clear infrared fluorescence was observed in a region where esophageal varices had been detected by conventional endoscopy when ICG was administered in doses of 0.005 mg/kg to 0.01 mg/kg, which was lower than the doses used in IRE. CONCLUSIONS: Compared with conventional IRE, IRFE facilitated the observation of deeper layers, and esophageal varices were observed by IRFE following the intravenous administration of a markedly reduced dose of ICG. IRFE, in combining the characteristics of reflected infrared light and fluorescence, may be a useful novel procedure in the diagnosis of vascular lesions in the gastrointestinal tract.

Basic study of an agent for reinforcement of near-infrared fluorescence on tumor tissue.

BACKGROUND AND AIMS: An indocyanine green derivative (ICG-sulfo-OSu) and agents for reinforcement of infrared fluorescence, which can be used as an infrared fluorescent labeling substance suitable for detection of microlesions by an IR fluorescence endoscope, have been developed. The study aims were to confirm the ability of a reinforcement agent, as well as imaging processing, to intensify fluorescence from the labeled antibody on immunohistochemical staining. SUBJECTS AND METHODS: ICG-sulfo-OSu-labeled MUC1 antibody and an IR fluorescence imaging system were employed in the present study. Paraffin sections of gastric cancer were stained with anti-MUC1 antibody by the avidin-biotinylated peroxidase complex method. Among the positive specimens, three cases were used for IR imaging analysis. Octylglucoside was used as a reinforcement agent. RESULTS: The incubation of paraffin sections with ICG-sulfo-OSu-labeled MUC1 antibody resulted in positive staining of the tumor sites by an IR fluorescence imaging system, and the intensity of fluorescence was increased depending on the concentration of octylglucoside and grade of imaging processing. CONCLUSION: A reinforcement agent, and image processing, intensify a labeled antibody excitable by infrared fluorescence in tumor sections and can generate a strong enough fluorescent signal to detect small cancers when examined with an infrared fluorescence endoscope.

Endoscopic sonography in the diagnosis of gallbladder wall lesions in patients with gallstones.

PURPOSE: The purpose of this study was to evaluate the diagnostic accuracy of endoscopic sonography (EUS) in the detection of gallbladder wall lesions in patients with and without gallstones. METHODS: We retrospectively reviewed the medical records, sonograms, and sonographic reports of 62 patients who underwent cholecystectomy for gallbladder wall lesions evaluated by EUS. We assessed the accuracy of EUS in diagnosing gallbladder wall lesions in the presence or absence of gallstones and on the basis of the size and number of stones and the size of the gallbladder wall lesions. We also evaluated the effect of acoustic shadowing. The EUS results were compared with the histopathologic results. RESULTS: EUS correctly diagnosed the gallbladder wall lesions in 17 (71%) of 24 patients with gallstones and in 34 (89%) of 38 patients without gallstones. The diagnostic accuracy of EUS was 86% in patients with gallbladder wall lesions smaller than 20 mm and 79% in patients with gallbladder wall lesions 20 mm or larger. The diagnostic accuracy was 75% in patients with gallstones smaller than 5 mm and 67% in patients with stones 5 mm or larger. The accuracy was 67% in patients with 1-5 stones and 83% in patients with 6 or more stones. None of these differences was statistically significant. Acoustic shadowing did not affect the diagnostic accuracy of EUS. CONCLUSIONS: The diagnostic accuracy of EUS for gallbladder wall lesions is not affected by the presence of gallstones. However, better diagnostic criteria must be established based on larger studies, and technical refinements of the equipment are needed to increase the accuracy of EUS in the diagnosis of gallbladder wall lesions.

Quantitative analysis of red color sign in the endoscopic evaluation of esophageal varices.

BACKGROUND AND STUDY AIMS: Bleeding due to esophageal variceal rupture is associated with an extremely high mortality rate. Variceal bleeding is frequent in patients who have a red color sign on endoscopy. However, the red color sign is subjectively evaluated on the basis of color tone and the shape of the varices. To allow standardization and facilitate consensus, an objective method of assessing the red color sign is needed. In this study, a system was established for quantifying the red color sign during endoscopic evaluation. PATIENTS AND METHODS: Between July 1995 and February 1997, 55 untreated patients with portal hypertension and esophageal varices identified on upper gastrointestinal endoscopy were enrolled in the study. Images obtained about 5 cm oral to the esophagogastric junction during endoscopy were stored on magnetic optical disks using an endoscopic image processor. The still images were transmitted to a computer and analyzed using computer software. The RGB components (R, red; G, green; B, blue) were measured at points showing flare consistent with the red color sign. The endoscopic assessment was based on the Japanese Research Society for Portal Hypertension's general rules for recording endoscopic findings in esophagogastric varices. RESULTS: The ratio of the red color area to the variceal area increased with increasing red color grade. There were significant positive correlations between the R and G, and G and B components. This suggests that comparing the R components alone would allow assessment of the color differences in the red color area and in the varices. The R value was significantly higher in the red color area (115 +/- 20) than in the varices (57 +/- 19). An R value of 90 was found at the boundary between the two parts (P < 0.001). CONCLUSIONS: The red color area can be automatically calculated and quantified using the analysis program. Improvements in data storage methods may allow real-time evaluation during endoscopy in the future.

Detection of human gastric cancer in resected specimens using a novel infrared fluorescent anti-human carcinoembryonic antigen antibody with an infrared fluorescence endoscope in vitro.

BACKGROUND AND STUDY AIMS: An indocyanine green derivative (ICG-sulfo-OSu) that can be used as an infrared fluorescent labeling substance suitable for detecting microlesions with an infrared fluorescence endoscope has been developed. The aims of the present study were to develop an infrared fluorescence endoscope and to demonstrate its usefulness in detecting cancerous tissue using an antibody coupled with ICG-sulfo-OSu. MATERIALS AND METHODS: ICG-sulfo-OSu-labeled mouse anti-human carcinoembryonic antigen (CEA) antibody and an infrared fluorescence endoscope were used in this study. Biopsy specimens of gastric cancer were stained with anti-CEA antibody using the avidin-biotinylated peroxidase complex method. The positive specimens used for the infrared imaging analysis were freshly resected stomachs from three patients. RESULTS: Treatment of freshly resected stomach specimens with ICG-sulfo-OSu-labeled-anti-CEA antibody complex resulted in positive staining of the tumor sites on infrared fluorescence endoscopy, and the infrared fluorescent images correlated well with the tumor sites. CONCLUSIONS: An anti-CEA antibody with affinity for cancerous lesions and labeled with ICG-sulfo-OSu can therefore be imaged using this infrared fluorescence endoscope. Specific antibodies tagged with ICG-sulfo-OSu can label cancer cells and can generate a strong enough fluorescent signal to detect small cancers when examined with an infrared fluorescence endoscope.

Relationship among gastric motility, autonomic activity, and portal hemodynamics in patients with liver cirrhosis.

BACKGROUND AND AIMS: We examined the effects of the autonomic nervous function and the volume of portal blood flow to clarify the mechanism of the abnormal gastric motility in patients with liver cirrhosis. METHODS: Heart rate variability, electrogastrogram (EGG), and volume of portal blood flow were measured before and after a meal in 27 patients with liver cirrhosis (LC group) and in 20 normal subjects (N group). Autonomic nervous function was evaluated by using spectral analysis of heart rate variability. We used the cine phase-contrast (PC) method, using magnetic resonance imaging (MRI) to measure the portal flow, while the peak frequency and spectral power of the EGG were measured at pre- and postprandial change. RESULTS: The ratio of low frequency power to high frequency power (LF/HF) was significantly higher, and the HF power was significantly lower in the LC group than in the N group both before and after a meal. In both groups, the electrogastrographic peak power ratio before and after a meal showed a positive correlation with the HF ratio, and an inverse correlation with the LF/HF ratio. In addition, portal blood flow volume was significantly decreased in the LC group than in the N group. However, the increased rate of portal blood flow after a meal correlated positively with the increased rate of electrogastrographic peak power. Moreover, gastric motility was positively correlated with esophageal varices and coma scale with the use of multivariate analysis. CONCLUSIONS: Parasympathetic hypofunction, sympathetic hyperfunction and portal hemodynamics were closely related with gastric motility in cirrhotic patients. In addition, gastric motility was decreased, at least in part, by the ingestion of food in cirrhotic patients because of abnormalities in autonomic functions and portal blood flow following a meal.

Asymptomatic case of congenital absence of the gallbladder.

Congenital absence of the gallbladder is rare among biliary abnormalities, and its preoperative diagnosis has been considered very difficult. We encountered a patient with congenital absence of the gallbladder and suggest a possible preoperative diagnosis of the abnormality, as well as reviewing the literature.

Vital immunostaining of human gastric and colorectal cancers grafted into nude mice: a preclinical assessment of a potential adjunct to videoendoscopy.

Videoendoscopy has not significantly advanced diagnostic accuracy beyond that attainable by conventional fiberscopy, with respect to microcarcinomas of the digestive tract. We suspected that after the labeling of these lesions with an agent detectable by videoendoscope, digital processing of the images could facilitate endoscopic diagnosis of microcarcinomas. We have developed a novel antibody labeled with an indocyanine green (ICG) derivative that is evident by videoendoscope. However, the binding of such an exogenous antibody in vivo to tumor surfaces has not been described. In this preliminary study, after transplanting human gastric cancer or colorectal cancer into nude mice, we successfully bound the tumors in vivo with an anti-MUC1 mucin antibody, as subsequently confirmed by the performing of immunohistochemistry with a secondary antibody. The antibody labeled with an ICG derivative may therefore be clinically useful in detecting gastrointestinal microcarcinoma by videoendoscopy.

Labeled carcinoembryonic antigen antibodies excitable by infrared rays: a novel diagnostic method for micro cancers in the digestive tract.

OBJECT: An indocyanine green derivative (ICG-sulfo-OSu) was used as the labeling substance for monoclonal antibody, and a fluorescence imaging system appropriate for ICG-sulfo-OSu excitable by infrared rays (IR) was developed. The goal of this study was to demonstrate antibody labeling at the tissue level using this new imaging system. MATERIALS AND METHODS: ICG-sulfo-OSu labeled mouse anti-human carcinoembryonic antigen (CEA) monoclonal antibody, a newly developed imaging system, and an infrared ray microscope were employed in this experiment. Paraffin sections of human colon cancer previously proven to have cross-reactivity to anti-CEA antibody were examined. RESULTS: Positive staining was seen as a brownish discoloration of oxidized 3,3'-diaminobenzidine tetrahydrochloride (DAB) in sections that reacted with ICG-sulfo-OSu-labeled anti-CEA antibody, and the fluorescence was well-matched with the oxidized DAB-positive sites. CONCLUSION: Specific antibodies labeled with ICG-sulfo-OSu have significant affinity to cancer cells and seem to reflect sufficient amounts of fluorescence by IR to be useful in a system for the endoscopic detection of micro cancers using the immunohistochemical staining method.

Endoscopic sonography in the diagnosis of xanthogranulomatous cholecystitis.

Xanthogranulomatous cholecystitis (XGC) is an unusual inflammatory disease of the gallbladder that may simulate gallbladder cancer. We report the findings with conventional sonography, endoscopic sonography (EUS), and CT in 3 cases of XGC. EUS could visualize hyperechoic nodules in the gallbladder wall, probably representing xanthogranulomas, but loss of the multilayered structure of the gallbladder wall and infiltration into adjacent organs make differentiating XGC from gallbladder cancer difficult with EUS alone.

The utility and limitations of an ultrasonic miniprobe in the staging of gastric cancer.

To determine the utility and limitations of an ultrasonic miniprobe (UMP) in the staging of gastric cancer, we evaluated 46 patients who underwent endoscopic ultrasonography (EUS) using an UMP and who were histologically determined to have gastric cancers. In every case, UMP findings were compared with histopathological findings after treatment. The total accuracy of UMP relative to the depth of tumor invasion was 71.7% (33/46 cases). Accuracy with respect to T1-m tumor diagnosis was 75.7% (22/29 cases), and for T1-sm, 76.9% (10/13 cases), but accuracy for T2 tumor diagnosis was low, due to ultrasound attenuation. When the analysis was carried out based on the size of tumor, the accuracy for UMP was 50.0% (9/18 cases) for all tumors over 20 mm and 85.7% (24/28 cases) for all tumors smaller than 20 mm. We conclude that UMP is suitable for investigation of tumor extension when the lesion is superficial and/or small gastric cancers which do not cause ultrasonic attenuation, but not when the tumor is large or located in certain sites, although conventional EUS is useful in some of these cases.

Vital immunohistochemical staining for a novel method of diagnosing micro-cancer. Examination of immunohistochemical staining of non-fixed fresh tissue.

It becomes possible to establish a novel diagnostic method for micro-cancer by modulating the signals from the lesion, if lesions can be labeled with substances which can be detected by video endoscopy. The authors have already succeeded in synthesizing indocyanine green (ICG) derivatives for a fluorescent labeling substance which emits near-infrared rays. Before the antibodies labeled by these substances can be used, it is necessary to establish a method of vital immunohistochemical staining. So, we investigated the responses of antibodies exposed to non-fixed fresh tissue specimens as a basic study on vital immunohistochemical staining. The responses of fresh esophageal and gastric specimens (biopsied or surgically resected) to immunohistochemical staining with anti-epithelial membrane antigen (EMA) antibodies under various conditions using the ABC method were examined. These tissue specimens were stained immunohistochemically, and incubated with anti-EMA antibodies for 10 and 30 minutes (esophagus), and for 60 and 120 minutes (stomach) at 37 degrees C. These results suggest that vital immunohistochemical staining is possible under optimum conditions. If an infrared fluorescent endoscopy catching this excited fluorescence can be developed, it will be possible to establish a new endoscopic diagnostic method on the basis of vital immunohistochemical staining.

Effect of administration of ursodeoxycholic acid at bedtime on cholesterol saturation of hepatic bile in Japanese patients with gallstone.

The administration of a single, daily 600 mg dose of ursodeoxycholic acid (UDCA) at bedtime and 3-200 mg doses per day at mealtime was conducted for 6 patients with gallstone and choledocholithiasis who were undergoing biliary drainage for the purpose of improving jaundice. Hepatic bile was collected from a drainage tube after a lapse of time in order to compare the bile acid compositions and cholesterol saturation index (SI) in bile for the 2 protocols. A significant increase in UDCA levels in hepatic bile was observed after both UDCA administration at bedtime and mealtime, but the effect of bedtime administration was significantly greater than that of mealtime administration. Whereas levels of cholic acid and chenodeoxycholic acid (CDCA) decreased for the case of bedtime administration, this was not detected for mealtime administration, although no significant differences among the mean interval values were observed. A significant in difference decreased SI was observed during UDCA bedtime administration, but not during mealtime administration, compared to the SI before administration. This suggests a decreased cholesterol excretion into the bile. Based on these findings and from the point of view of compliance, bedtime administration of UDCA appears to be an effective method.

Antibodies labeled with fluorescence-agent excitable by infrared rays.

Endoscopy is not significantly better than fiberoscopy for the diagnosis of minute cancers of the digestive tract. However, labeling of these lesions with an agent that can be detected videoendoscopically, with subsequent computer processing of the electronic signals, should facilitate endoscopic diagnosis of microlesions. We developed an antibody labeled with an indocyanine green(ICG) derivative that has a specific fluorescence emission at 807 nm upon excitation at 768 nm. The physiochemical characteristics of this labeled antibody resemble those of ICG. The activity of the antibody is suitable for immunohistochemical staining, and the antibody fluoresces under infrared ray excitation. This antibody should prove useful for performing vital immunostaining for infrared endoscopy.