Virtual histology of colorectal lesions using laser-scanning confocal microscopy

BACKGROUND AND STUDY AIMS: Histological examination of gastrointestinal lesions is currently based on light-microscopic examination of thin-slice specimens, with hematoxylin and eosin staining. A study of the use of laser-scanning confocal microscopy (LCM) to obtain immediate microscopic images of untreated specimens for examining colorectal lesions was carried out. A probe-type LCM prototype endomicroscope that can be passed through the working channel of an endoscope has also been developed. MATERIALS AND METHODS: The study materials consisted of colorectal lesions resected either endoscopically or surgically at Showa University Northern Yokohama Hospital. One hundred untreated specimens were examined using LCM. The histopathological findings in the lesions were seven cases of normal colonic mucosa, five hyperplastic polyps, 68 adenomas with low-grade dysplasia, 10 adenomas with high-grade dysplasia, and 10 adenocarcinomas. An argon laser beam with a wavelength of 488 nm was used for the LCM study. Observation of the resected normal colonic mucosa (in vitro) and the rectal mucosa of a healthy volunteer (in vivo) was possible using the endomicroscope. The LCM images for each specimen were compared with the hematoxylin-eosin-stained histopathological cross-sections. RESULTS: The LCM images corresponded well with the conventional hematoxylin-eosin light-microscopic images. The nuclei were not visualized in normal mucosa or hyperplastic polyps. In adenomas with high-grade dysplasia and carcinomas, nuclei were more often visible than in adenomas with low-grade dysplasia. The rate of visualization of nuclei was significantly different ( P < 0.01) between these two groups (60.0 % vs. 10.3 %). In LCM images using endomicroscope, it was possible to recognize the orifices of the colonic glands and goblet cells both in vitro and in vivo. CONCLUSIONS: Laser-scanning confocal microscopy provides immediate images that correspond well with those of hematoxylin-eosin staining. An improved probe-type LCM endomicroscope is being developed which should provide better histological images of colorectal lesions in vivo.     

Validation of the quality of histological images obtained of fresh and formalin-fixed specimens of esophageal and gastric mucosa by laser-scanning confocal microscopy

BACKGROUND AND STUDY AIMS: We have previously reported the success of a method of virtual histology using laser-scanning confocal microscopy (LCM) in vitro on untreated fresh specimens obtained from the gastrointestinal mucosa. In the present study, we aimed to apply LCM to both fresh and formalin-fixed specimens, without additional treatment, in order to validate and compare the quality of the images obtained. METHODS: We obtained 18 specimens from 11 patients, either by endoscopic biopsy or following surgical resection. First, we observed the fresh, saline-immersed specimen with LCM using the Fluroview microscope (Olympus Co. Ltd., Tokyo, Japan). We then fixed the specimen with formalin and obtained further LCM images 1 hour, 3 hours, and 24 hours after fixation. Three independent observers observed the images and were asked to assess the origin of the samples, the treatment of the samples, the time after formalin fixation, and whether they showed benign or malignant lesions. We used kappa statistics to compare the agreement among the three observers in each of these four areas of interest. RESULTS: Between January and March 2003, we obtained 191 LCM images from 18 specimens. Thirty images were randomly selected for observation. The overall accuracy for differentiating between esophagus and stomach specimens was 96.6 %. The accuracy of differentiating normal from cancerous lesions was 92.2 %. The differentiation between saline-immersed and formalin-fixed specimens was 59.7 % accurate and the assessment of the time interval after formalin fixation was only 37.3 % accurate. The kappa statistics showed that there was strong interobserver agreement on the differentiation of specimen origin and of cancerous from benign lesions. However, there was no agreement among the observers on the method of specimen preparation or on the estimated time interval after formalin fixation. CONCLUSIONS: We concluded that images obtained from fresh specimens using LCM were of a quality good enough to make an accurate diagnosis of upper gastrointestinal carcinoma.     

In vivo observation of living cancer cells in the esophagus, stomach, and colon using catheter-type contact endoscope, "Endo-Cytoscopy system"

A catheter-type endomicroscope has been developed with a maximum magnifying power of 1100 times. Living cancer cells in the esophagus, stomach, and colon were successfully observed in high-resolution images. The "Endo-Cytoscopy system" (prototype,Olympus Optical, Co., Tokyo, Japan) is a catheter-based probe capable of passage through the accessory channel of the endoscope(GIF-1T, Olympus). Methylene blue solution was used for vital staining of the in vivo gastrointestinal mucosa. Living cells in both normal mucosa and malignant tissue were clearly demonstrated in luminal organs. In particular, the nucleus, cell body, and nucleolus were clearly demonstrated with high-quality images similar to those of conventional cytology. This novel technology has the potential to provide an in vivo histologic diagnosis via "optical biopsy" and virtual histology.     

Optical coherence tomography in the gastrointestinal tract

Conventional gastrointestinal endoscopic instruments have provided ready access to the mucosal surface of the esophagus, stomach,small intestinal, colon, bile duct, and pancreatic duct. The evolution of endoscopic ultrasonography has provided an additional dimension to the clinical application of modern endoscopy and imaging technology.     

In vivo endoscopic optical coherence tomography of esophagitis, Barrett's esophagus, and adenocarcinoma of the esophagus

BACKGROUND AND STUDY AIMS: We studied the feasibility of endoscopic optical coherence tomography imaging in esophageal disorders, including Barrett's esophagus and Barrett-related adenocarcinoma. Optical coherence tomography is a high-resolution cross-sectional imaging technique with a resolution of almost 10 microm. PATIENTS AND METHODS: The mucosal architecture of reflux esophagitis (n = 9) and Barrett's esophagus (n = 9) including Barrett-related esophageal cancer (n = 6) was studied by optical coherence tomography imaging. RESULTS: In different stages of reflux esophagitis edema, fibrinoid deposits, or loss of the epithelial layer were observed. Optical coherence tomography images of Barrett's esophagus substantially differed from normal esophagus, reflux esophagitis, and esophageal carcinoma. A stratified structure of the mucosa was still preserved in Barrett's esophagus. However, images of Barrett-related cancer lacked the regular structure of the esophagus. CONCLUSIONS: The high consistency of the first optical coherence tomography findings, the resolution of up to 10 microm, and the distinct pattern of normal, inflammatory, premalignant and malignant tissues make optical coherence tomography a promising method for endoscopically obtained optical biopsy.     

Enhanced gastrointestinal diagnosis: light-scattering spectroscopy and optical coherence tomography

Detection of microscopic and biochemical changes within the mucosa and submucosa are beyond the realm of routine endoscopy. Distinguishing hyperplastic from neoplastic polyps, differentiating malignant from benign ulcers, and detecting mucosal dysplasia in patients with ulcerative colitis or Barrett's esophagus remain within the purview of the gastrointestinal pathologist. Recent developments in tissue spectroscopy and endoscopic optical coherence tomography have the potential to expand significantly our ability to diagnose gastrointestinal disease beyond the capabilities of visible light endoscopy.     

结肠癌组织中p53、p14基因表达的研究

目的　探讨结肠癌组织中 p5 3、p14基因的表达及其与结肠癌某些生物学行为的关系。 方法　经病理诊断明确的内镜活检组织标本 318例 ,其中癌组织 12 6例 ,腺瘤组织 84例 ,正常组织 10 8例。采用免疫组织化学S -P法检测三种组织中p5 3、p14的表达。 结果　p5 3在结肠癌组织中的表达明显高于正常组织 (P<0 .0 5 ) ,p14在腺瘤和癌组织中的表达低于正常结肠组织 (P <0 .0 5 ) ,p14与癌组织的临床分期及组织分化程度相关 (P >0 .0 5 ) ,p5 3与 p14的表达存在负相关性 (r =- 0 .816 9)。 结论　p5 3、p14基因的表达异常可能与结肠癌的发生发展有关 ,联合检测两者的协同表达可作为结肠癌辅助诊断的参考指标。     

Rapid development of an advanced squamous-cell carcinoma of the esophagus

This report describes the case of a 42-year-old alcoholic man who developed advanced squamous-cell carcinoma of the esophagus following an endoscopic examination showing grossly normal mucosa only 8 months previously. We believe this is the first case report providing endoscopic images illustrating a progression from grossly normal mucosa to advanced carcinoma of the esophagus in only 8 months.     

Endoscopic circumferential esophageal mucosectomy in a porcine model: an assessment of technical feasibility, safety, and outcome

BACKGROUND AND STUDY AIMS: Endoscopic mucosectomy has been performed for early cancers and dysplastic lesions < or = 2 cm in diameter. The feasibility and safety of mucosectomy for circumferential lesions of the esophagus is uncertain. The aim of this study was to determine the technical feasibility, as well as the short and long-term complication rates, with circumferential endoscopic mucosectomy of the distal esophagus in the pig. MATERIALS AND METHODS: Circumferential endoscopic mucosectomy of the distal 3 cm of the esophagus was performed in four pigs, using a cap mucosectomy device. The animals were sacrificed after 30, 50, 70, and 90 days to assess mucosal regeneration and stricture formation. RESULTS: The procedure time for circumferential endoscopic mucosectomy was 15-30 min. Circumferential endoscopic mucosectomy was technically feasible and without short-term complications. Videotapes of all resections were reviewed to ensure that complete removal of the mucosa was achieved. All mucosectomy specimens underwent histological evaluation. The specimens included the mucosa alone in three of the pigs. Some of the specimens in the fourth pig included a superficial layer of muscularis propria. This pig failed to thrive. Macroscopic examination of the dissected esophageal specimens from the healthy pigs revealed a well-healed, normal-appearing esophagus, whereas a stenosis of 4 x 10 mm was observed in the distal esophagus of the pig that failed to thrive. CONCLUSIONS: Circumferential endoscopic mucosectomy of the porcine distal esophagus is feasible and safe. An adequate submucosal saline cushion is essential to prevent stenosis due to deep injury.     

Biopsy methods and pathology of Barrett's esophagus

We reviewed the definition of the esophagogastric junction and the biopsy sites and histologic findings of biopsy specimens from Barrett's esophagus. The borderline between the esophagus and stomach has been defined as the distal limit of the longitudinal vessels by the Japan Esophageal Society, because the longitudinal vessels are always located within the esophagus. As squamous islands in Barrett's mucosa are usually the orifices of esophageal glands proper, biopsy specimens from the squamous islands show esophageal glands proper or their ducts. The identification of esophageal glands proper is a definite histological indicator that a piece of biopsy tissue is of esophageal origin. Therefore, a diagnosis of Barrett's esophagus can be made purely on the basis of the histologic findings in these biopsy specimens of squamous islands. Since columnar mucosa is usually recognizable at endoscopy, a diagnosis of Barrett's esophagus can be made solely on the basis of endoscopic examination, without any need for histologic confirmation, if squamous islands are recognized in columnar-lined mucosa.     

Diagnosis of Barrett's esophagus by pertechnetate radionuclide.

In Barrett's esophagus the normal stratified squamous epithelium of esophagus is replaced by columnar epithelium and other charcteristics of gastric mucosa. Barrett's esophagus has an increased tendency to bleed and is more prone to undergo malignant change. There are many procedures used to diagnose this entity, but only by serial and multiple esophageal mucosal biopsies can the diagnosis be confirmed. Harper et al (2) demonstrated an early and intense uptake of 99m Tc pertechnetate by the stomach in animals. Since the Barrett's esophagus is lined by gastric mucosa, pertechnetate scintigraphy was used as a screening procedure. The criteria for a postive scan was an area of increased uptake of technetium extending above the normal dense uptake of stomach configuration. Pertechnetate scintigraphy was performed in 4 patients with Barrett's esophagus and 6 controls with only one false negative result. Thus pertechnetate scintigraphy is a rapid, safe, and atraumatic screening procedure.     

Histamine metabolism of the human gastric mucosa--a study on the regional distribution of the amine and enzyme activities

Histamine content, histidine decarboxylase activity and histamine methyl transferase activity of the human gastric mucosa have been studied in healthy subjects. Histamine content was evenly distributed over the gastric mucosa and no significant difference could be observed between the oxyntic and pyloric gland area. A 'specific' histidine decarboxylase was found in the human gastric mucosa and the enzyme activity was significantly higher in the corpus compared to the antral mucosa. We found no difference in enzyme activity of the mucosa obtained from three defined areas of the corpus region of the stomach. Histamine methyl transferase activity was of the same magnitude in the corpus as in the antral mucosa. In addition, with regard to histamine methyl transferase activity, we were unable to demonstrate any difference in activity in the mucosal specimens obtained from the defined areas of the stomach. The presented technique to obtain gastric mucosal tissue specimens during routine fibreoptic endoscopy of the stomach, allows studies over gastric mucosal histamine metabolism in health as well as in disease states.     

Endoscopic examination including magnifying endoscopy for diagnosis of GERD

Symptoms of heartburn and regurgitation are the most important for diagnosis of gastroesophageal reflux disease (GERD) in the clinical field. Endoscopic examination is also widely used modality for Los Angeles classification of GERD according to endoscopic severity of esophageal mucosal breaks. However, about half of GERD patients reveal no abnormality under conventional endoscopy. These endoscopic negative GERD is called as non-erosive reflux disease (NERD). There is the possibility to underestimate a minute mucosal change of GERD by conventional endoscopy that has the limitation of visual ability. Magnifying endoscopic examination is able to get clear visualization of intrapapillary capillary loops(IPCL), which are usually shown as dot-like structures in esophageal mucosa by a conventional endoscopy. The changing of IPCLs is associated with inflammation and neoplasia of esophagus. Minute change of IPCLs such as a dilation and elongation with regular intervals were reported to be suggestive of inflammatory change in esophagus. Magnifying endoscopic observation of IPCLs is useful for diagnosis of NERD which cannot be visualized by conventional endoscopy.     

Circumferential endoscopic mucosal resection in Barrett's esophagus with high-grade intraepithelial neoplasia or mucosal cancer. Preliminary results in 21 patients

BACKGROUND AND STUDY AIMS: Treatment by endoscopic mucosal resection (EMR) has been established for early lesions in Barrett's esophagus. However, the remaining Barrett's esophagus epithelium remains at risk of developing further lesions. The aim of this study was to evaluate the efficacy of circumferential endoscopic mucosectomy (circumferential EMR)s in removing not only the index lesion (high-grade intraepithelial neoplasia (HGIN) or mucosal cancer), but also the remaining Barrett's esophagus epithelium. PATIENTS AND METHODS: A total of 21 patients were included in the study (11 men, 10 women), who had Barrett's esophagus and either HGIN (n = 12) or mucosal cancer (n = 9). Of the patients, 17/21 were at high surgical risk and five had refused surgery. On the basis of preprocedure endosonography their lesions were classified as T1N0 (n = 19) or T0N0 (n = 2). The lesions and the Barrett's esophagus epithelium were removed by polypectomy after submucosal injection of 10-15 ml of saline; a double-channel endoscope was used in 15/21 cases. Circumferential EMR was performed in two sessions, the lesion and the surrounding half of the circumferential Barrett's esophagus mucosa being removed in the first session. In order to prevent the formation of esophageal stenosis, the second half of the Barrett's esophagus mucosa was resected 1 month later. RESULTS: Complications occurred in 4/21 patients (19 %), consisting of bleeding which was successfully managed by endoscopic hemostasis in all cases. No strictures were observed during follow-up (mean duration 18 months) and endoscopic resection was considered complete in 18/21 patients (86 %). For three patients, histological examination showed incomplete removal of tumor: one of these underwent surgery; two received chemoradiotherapy, and showed no evidence of residual tumor at 18 months' and 24 months' follow-up, respectively. Two patients in whom resection was initially classified as complete later presented with local recurrence and were treated again by EMR. Barrett's esophagus mucosa was completely replaced by squamous cell epithelium in 15/20 patients (75 %). CONCLUSIONS: Circumferential EMR is a noninvasive treatment of Barrett's esophagus with HGIN or mucosal cancer, with a low complication rate and good short-term clinical efficacy. Further studies should focus on long-term results and on technical improvements.     

Correlation of in vitro autofluorescence endoscopy images with histopathologic findings in stomach cancer

BACKGROUND AND STUDY AIMS: There is increasing interest in the use of autofluorescence endoscopic imaging systems for the detection of malignancies in the gastrointestinal tract. The purpose of this study was to investigate the autofluorescent color of cancer lesions, and the detection rate of cancer areas using an autofluorescence endoscopic imaging system. Concurrent histopathologic examination was used for comparison. PATIENTS AND METHODS: This study involved a total of 50 stomach cancer patients (with a total of 61 lesions) undergoing surgery. Immediately after the resection, each lesion was observed using an autofluorescence endoscopic imaging system (LIFE-GI system, Light-Induced Fluorescence Endoscopy in the Gastrointestinal Tract). A total of 429 evaluation points in the specimens were selected according to tumor size. The images obtained in this way were assessed by comparison with histopathologic findings in terms of depth of invasion, thickness of the mucosa that had been invaded by cancer cells, and the histologic type of each cancer. RESULTS: Detection with the LIFE-GI system was possible in 58 of the 61 stomach cancer lesions (95.1%). When examined in detail with reference to sampling points, the detection rates of cancer areas according to depth of cancer cell invasion were 57.5 % for invasion to the mucosa, 74.3 % for invasion to the submucosa, and 88.1% for invasion to the muscularis propria or deeper. The detection rates increased as the depth of invasion increased, and they increased significantly as the mucosa invaded became thicker. Detection rates according to histologic type were 82% for differentiated cancer, and 61% for undifferentiated cancer. Most of the cancer areas appeared dark red. Light blue, brilliant red, and white areas were also observed. CONCLUSIONS: Mucosal thickening as a result of cancer cell invasion had a large impact on the detection rate of the LIFE-GI system. As a result, this method may not be useful for the detection of undifferentiated cancer, which may invade in a more dispersed manner, without altering the mucosal thickness.     

Endoscopic mucosal resection scissors for the treatment of early gastric cancer

Endoscopic mucosal resection (EMR) is currently a common treatment for superficial gastrointestinal tumors. We have developed new EMR scissors for superficial lesions in the esophagus and stomach. These scissors have stainless steel blades with an electrocoagulation device for hemostasis. We report a case in which superficial gastric cancer was treated by means of the EMR scissors. The lesion was removed using the scissors and no major complication was encountered. EMR scissors can be used for endoscopic resection of superficial lesions of the esophagus and stomach.     

Squamous reepithelialization after circumferential endoscopic mucosal resection of superficial carcinoma arising in Barrett's esophagus

Recent reports on the results of endoscopic ablation of Barrett's mucosa have been promising, particularly when total mucosal ablation is coupled with aggressive acid-suppression treatment using high-dose proton-pump inhibitor therapy. There is also a considerable literature on reepithelialization after ablative treatments in Barrett's esophagus. This report describes a case of multifocal superficial adenocarcinoma arising in Barrett's mucosa that was successfully treated with total circumferential endoscopic mucosal resection, with a subsequent follow-up of more than 2 years. This is the first report describing the process of squamous reepithelialization after endoscopic mucosal resection in Barrett's esophagus.     

Upper gastrointestinal tract lesions in patients with nonspecific ulcerative colitis

Endoscopic and morphological examinations of the upper gastrointestinal tract (GIT) were made in 80 patients with nonspecific ulcerative colitis (NUC). Tests for Helicobacter pylori infection and gastric juice acidity were also conducted. It was found that in many patients NUC was associated with development of nonspecific esophagitis, gastritis and duodenitis. The endoscopic examination revealed erosions of the abdominal esophagus in 13.8% cases, erosive-ulcerative alterations in the stomach and duodenum in 27.5% of NUC patients. The most marked changes occurred in patients with severe NUC. Alterations in the upper GIT were not related with either Hp or the acidity. Morphological examinations of the biopsies from the esophagus, stomach and duodenum demonstrated changes similar to those in biopsy material from the colon. The same type of morphological changes in mucosa from the upper GIT and the colon in NUC indicates that their pathogenesis is also the same.     

In Vitro Ultrasound Biomicroscopic Imaging of Colitis in Rats

Objective. The purpose of this study was to show the feasibility of 50‐MHz ultrasound biomicroscopy (UBM) to image the rat colon. Methods. B‐mode images were obtained from ex vivo colon samples (n = 4) collected from Rattus norvegicus (Berkenhout, 1769) rats, with 2,4,6‐trinitrobenzene sulfonic acid–induced colitis in 3 of them. Left colon rectangular fragments (5 × 5 mm) were obtained after necropsy, and UBM images were acquired with the samples immersed in saline at 37°C. All layers of the normal intestinal wall were analyzed according to their thickness and the presence of uneven bowel mucosa (ulcers). The folds and layers detected by UBM were correlated with histopathologic analysis. Results. The 4 layers of the normal colon were identified on the UBM images: the mucosa (hyperechoic), muscularis mucosae (hypoechoic), submucosa (hyperechoic), and muscularis externa (hypoechoic). On 2 UBM images, superficial ulcers were detected, approximately 0.5 mm in size, with intestinal involvement limited to the mucosa. The histopathologic analysis verified enlargement of submucosa layers due to an edema associated with sub‐mucosa leukocyte infiltration. On 1 UBM image, it was possible to detect a deep ulcer, which was confirmed by the light microscopic analysis. Conclusions. An ultrasound imaging system was scaled and optimized to visualize the rat colon. Ultrasound biomicroscopy provided axial and lateral resolutions close to 25 and 45 μm, respectively, and adequate penetration depth to visualize the whole thickness of an inflamed colon. The system identified the colon layers and was able to detect mural changes and superficial ulcers on the order of 500 μm.     

Two-dimensional computer color graphics of gastric mucosal blood distribution in normal subjects and ulcer patients.

The mucosal blood volume in 20 to 24 different regions of stomach was estimated by reflectance spectrophotometry during endoscopy, and an image showing mucosal blood distribution was made by two-dimensional computer color graphics with the aid of a personal computer. In 55 normal controls, the estimated mucosal blood volume (EMBV) was greater in the corpus mucosa than in the antral mucosa, and less in the lesser curvature than in the greater curvature. The volume in the anterior and posterior walls was almost the same. In 15 patients with active gastric ulcers in the angular region, the EMBV was decreased in all regions in the stomach. In 37 patients with healing ulcers, the EMBV increased, resuming the same levels as in normal controls. However, the EMBV around the ulcer increased remarkably at this stage. In 35 patients with ulcers in the scarring stage, the distribution of the EMBV was similar to that in the normal controls. These hemodynamic changes were shown clearly in a color display with the aid of a personal computer. This method could offer new possibilities in endoscopic research.