Yoshihiro Kusaka

Basic studies on a labeled anti-mucin antibody detectable by infrared-fluorescence endoscopy

Background. We developed a fluorescent dye, indocyanine green (ICG)-sulfo-OSu, which was excited by infrared rays and conjugated to various antibodies. We attempted to clarify the staining patterns of anti-sulfomucin and anti-MUC1 antibodies in gastrointestinal cancer. We then evaluated the potential of the dye as a fluorescent label for antibodies specific to cancer, to be used as a diagnostic method for microcancer, with infrared fluorescence endoscopy. Methods. Paraffin sections of samples collected from 10 patients with esophageal cancer, 30 patients with gastric cancer, and 20 patients with colorectal cancer were immunohistologically stained using an anti-sulfomucin antibody and an anti-MUC1 antibody, and the staining patterns were examined. If a section had a high staining intensity, it was reacted with the ICG-suflo-OSu-labeled antibody and evaluated with infrared fluorescence imaging. Results. The staining patterns with the antibodies varied depending on the organs and the histological types and depth of the cancers, but the staining was generally good and the staining on the mucosal surface of cancer tissues was retained. Good images of cancer cells could be obtained by infrared fluorescence observation using the ICG-sulfo-OSu-labeled anti-MUC1 antibody. Conclusions. The anti-MUC1 antibody stained gastrointestinal cancer cells well, and nearly specific infrared fluorescence in cancer tissues was observed using the labeled anti-MUC1 antibody. The ICG-sulfo-OSu-labeled anti-MUC1 antibody has possible usefulness for the screening of cancer via infrared fluorescence endoscopy.

A new infrared fluorescent-labeling agent and labeled antibody for diagnosing microcancers.

PURPOSE We have developed infrared fluorescent labeling agents and infrared-ray fluorescence endoscopes to establish a novel diagnostic technique. Since the fluorescence intensity of the initial labeled antibody (ICG-sulfo-OSu-labeled antibody) was not sufficient for practical use, we synthesized indocyanine green acylthiazolidinethione (ICG-ATT), which was expected to label various target molecules having amino groups efficiently. MATERIALS AND METHODS To confirm imaging of infrared fluorescence intensity of ICG-ATT- and ICG-sulfo-OSu-labeled anti-MUC1 antibodies, cotton thread was soaked in various concentrations of the antibody solution in 0.1M PBS, and observed under the epi-illumination infrared fluorescence microscope. Localization and the intensity of infrared fluorescence and DAB coloring was compared in paraffin sections of human gastric mucosa. RESULTS In the study of cotton threads, both labeled antibodies showed relatively clear infrared fluorescence, and significant difference was not observed between the two antibodies. ICG-ATT-labeled anti-MUC1 antibody produced stronger staining than that by ICG-sulfo-OSu-labeled antibody. Localization pattern of infrared fluorescent staining was in good agreement with that by the conventional method with oxidized DAB staining. CONCLUSION ICG-ATT is useful as a fluorescent-labeling agent for diagnosis of microcancers by infrared fluorescence endoscopes.

Reflected illumination-type imaging system for the development of infrared fluorescence endoscopy

Fluorescent labeled monoclonal antibodies against carcinoembryonic antigen (CEA) may provide a specific label for lesions of the GI tract. We previously developed an indocyanine green (ICG) derivative (ICG N‐hydroxy sulfo succinimide ester; ICG‐sulfo‐Osu) as a fluorescent label that is excited by infrared rays and is suitable for vital immunohistochemical staining. We also previously developed a transmitted illumination‐type (transmitted type) fluorescence imaging system that uses infrared rays to detect ICG‐sulfo‐Osu. In this paper we describe a new reflected illumination‐type (reflected type) imaging system for infrared fluorescence endoscopy. We tested the efficacy of this system on sections of human esophagus and normal skeletal muscle stained with ICG‐sulfo‐Osu labeled anti‐epithelial membrane antigen (EMA) antibody, and on sections of human gastric cancer tissue stained with ICG‐sulfo‐Osu labeled anti‐CEA antibody. Infrared fluorescent images were obtained with both fluorescent antibodies, and results correlated well with oxidized DAB‐positive sites. Vital immunohistochemical staining of micro cancers should be detectable by exciting an ICG‐sulfo‐Osu labeled antibody specific to the tumor cells with infrared rays, using this reflected type imaging system.

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

Abstract: 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.

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.

7172 Endoscopic ultrasonography for the diagnosis of gallbladder lesions in patients with gallstones.

Purpose: Gallstones are generally considered a risk factor for gallbladder cancer. Since gallbladder cancer has a poor prognosis, accurate qualitative diagnosis of elevated lesions in the gallbladder is needed, even in the presence of gallstones. In this study, the ability of endoscopic ultrasonography (EUS) to detect gallbladder lesions in patients with gallstones was assessed. Patients and Methods: Between April 1992 and September 1999, 62 patients underwent cholecystectomy for an elevated lesion or thickening of the gallbladder wall identified by imaging techniques including EUS. EUS was performed using the Olympus GF-UM20 (7.5MHz) system. The accuracy of EUS was analyzed in relation to the presence or absence of gallstones, the size and number of stones, and presence or absence of acoustic shadowing.The EUS results were compared with histopathologic results. Significance was tested using the chi-square test and the Students t test. Results: The accuracy of EUS was 70.8% in the gallstone group and 89.5% in the gallstone-free group (NS). The diagnostic accuracy was 75.0% in the patients with stones smaller than 5 mm, 33.3% in the patients with stones between 6 and 10 mm in size, and 77.8% in the patients with stones larger than 11 mm (NS). The accuracy was 66.7% in the patients with 1 to 5 stones, and 83.3% in the patients with 6 or more stones (NS). Acoustic shadowing did not affect the diagnostic accuracy of EUS. Conclusions: Gallstones do not affect the diagnostic accuracy of EUS for gallbladder lesions. However, more diagnostic criteria must be established, and new devices need to be introduced that can provide more information about the lesions.

7128 Three-dimensional ct pancreatography (3d-ctp) of pancreatic diseases.

Background: Endoscopic retrograde pancreatograpy (ERP) is common techniques used for the diagnosis of pancreatic diseases. However, conventional ERP under fluoroscopy (c-ERP) provides only two-dimensional projection images of the pancreatic ducts. Recent advances in the volumetric CT enable us to get whole pancreatic deta. CT images provide better tissue characterization than fluoloscopy images and ability to create 3D images from its data. Using volumetric CT technique with ERP is thought to be able to provide additional information. Aims: The present study was undertaken to assess the usefulness of 3D-CTP in the diagnosis of pancreatic diseases. Methods: The subjects of this study were 22 patients with pancreatic disease (5 with pancreatic cancer, 5 with chronic pancreatitis and 12 with pancreatic cystic disease). After c-ERP with balloon-catheter, 3D-CTP was performed with volumetric CT within a single brethhold period. Several 3D images per a case were made with the method of shaded surface display(SSD), multiplanar reconstruction (MPR) and maximum intensity projection (MIP) from CT data. Results: Images with SSD and MIP allowed us to realize the pancreatic ducts three dimensionally. The main pancreatic duct (MPD) was visualized three dimensionaly, but the side branches were not enough (see table).Images with MPR provide good visualization of both the pancreatic duct and its parenchyma. In the case of pancreatic cyst, the communication between the cyst and MPD was detected with 3D-CTP. In the case of mutin producing tumor, papillary intraductal tumor was detected more clearly. In the case of pancreatic cancer, dilated pancreatic ducts proximal to the stenosis were observed by 3D-CTP, which could not be visualized by c-ERP, with advantage of contrast resolution of CT. Conclusion: 3D-CTP provides three dimensional images of pancreatic ducts and its parenchyma. It provides additional information about the pancreatic pathologies to c-ERP. 3D-CTP with c-ERP seems to be applicable to simulation for the surgery. The accuracy and clinical usefulness of 3D-CTP will elevated by using improved devices under optimal condition.