Technology insight: Laser-scanning confocal microscopy and endocytoscopy for cellular observation of the gastrointestinal tract

Recent advances in endoscopic imaging technology have enabled the visualization of early-stage cancer and its precursors in the gastrointestinal tract. Chromoendoscopy, magnifying endoscopy, endoscopic optical coherent tomography, spectroscopy, and various combinations of these technologies, are all important for the recognition of small and unclear lesions. To observe cancer cells in vivo, two types of ultra-high magnifying endoscope--'laser-scanning confocal endoscopy series' and 'contact endoscopy series'--that have a maximum of more than 1,000x magnifying power have been developed. These endoscopes can generate high-quality images of both living cancer cells and normal cells in the gastrointestinal tract, with a quality comparable to that possible with conventional cytology. These novel imaging technologies may make in vivo histological diagnosis by virtual histology possible.     

Novel endoscopic imaging techniques toward in vivo observation of living cancer cells in the gastrointestinal tract

Recent advances in endoscopic imaging technology have enabled the visualization of cellular-level microstructures of early-stage cancer and its precursors. Chromoendoscopy, magnifying endoscopy, endoscopic optical coherent tomography (EOCT) spectroscopy, and various combinations of these technologies, are all important for the recognition of small and unclear lesions. In order to observe cancer cells in vivo two types of ultra-high magnifying endoscope--'laser-scanning confocal laser-scanning endoscopy series' and 'contact endoscopy series'--that have a maximum of >1,000 times magnifying power have been developed. The use of these endoscopes has allowed the generation of high quality images of both living cancer cells and normal cells in the gastrointestinal tract. In particular, clear images of cells and their nuclei, equivalent to the high quality that is possible with conventional cytology, have been produced. These novel imaging technologies may make in vivo histological diagnosis by virtual histology possible.     

Endoscopic imaging in Barrett’s esophagus

Barrett’s esophagus is the only known precursor that predisposes patients to the development of esophageal adenocarcinoma. The current recommended surveillance method is targeted biopsies of any abnormalities followed by random four-quadrant biopsies every 2 cm using standard white light endoscopy. Compliance with this and sampling error are two of the biggest problems. Several novel imaging technologies have been developed to aid the diagnosis of early neoplasia in Barrett’s esophagus. There are emerging data that some of these new modalities can increase the yield of detecting dysplasia. This review will discuss some of the present available techniques and technologies including chromoendoscopy, narrow-band imaging, autofluorescence imaging, optical coherence tomography, confocal endomicroscopy and endocytoscopy. Based on the current evidence, these imaging modalities appear to be promising as adjunctive tools to white light endoscopy. A few of them, nevertheless, remain experimental due to expense, lack of expertise, generalizability as well as reproducibility of results.     

Photodiagnostic techniques for the endoscopic detection of premalignant gastrointestinal lesions

Considerable attention is given to the clinical diagnosis of gastrointestinal (GI) malignancies as they remain the second leading cause of cancer‐associated deaths in developed countries. Detection and intervention at an early stage of preneoplastic development significantly improve patient survival. High‐risk assessment of asymptomatic patients is currently performed by strict endoscopic surveillance biopsy protocols aimed at early detection of dysplasia and malignancy. However, poor sensitivity associated with frequent surveillance programs incorporating conventional screening tools, such as white light endoscopy and multiple random biopsy, is a significant limitation. Recent advances in biomedical optics are illuminating new ways to detect premalignant lesions of the GI tract with endoscopy. The present review presents a summary report on the newest developments in modern GI endoscopy, which are based on novel optical endoscopic techniques: fluorescence endoscopic imaging and spectroscopy, Raman spectroscopy, light scattering spectroscopy, optical coherence tomography, chromoendoscopy, confocal fluorescence endoscopy and immunofluorescence endoscopy. Relying on the interaction of light with tissue, these ‘state‐of‐the‐art’ techniques potentially offer an improved strategy for diagnosis of early mucosal lesions by facilitating targeted excisional biopsies. Furthermore, the prospects of real‐time ‘optical biopsy’ and improved staging of lesions may significantly enhance the endoscopist's ability to detect subtle preneoplastic mucosal changes and lead to curative endoscopic ablation of these lesions. Such advancements within this specialty will be rewarded in the long term with improved patient survival and quality of life.     

New techniques in imaging in Barrett's esophagus

The presence of dysplasia in patients with Barrett's esophagus identifies who is at increased risk for the development of esophageal adenocarcinoma and who may most benefit from intervention. Several technologies have emerged as potent tools to identify subtle or occult neoplasia in the gastrointestinal tract. Detailed inspection of the mucosa with high resolution white light endoscopy is the most critical tool to detect subtle neoplasia. This review also chromoendoscopy, narrow band imaging, autofluorescence imaging, optical coherence tomography, confocal laser endomicroscopy, and spectroscopy in the context of detection of neoplasia in Barrett's esophagus.     

DIAGNOSIS OF BARRETT’S ESOPHAGUS AND ESOPHAGEAL NEOPLASIA: EAST MEETS WEST

Barrett’s esophagus has traditionally been considered to be a predominantly ‘Western world’ neoplastic condition. However, over the years, Asian countries are beginning to diagnose increasing numbers of patients with gastroesophageal reflux disease, columnar metaplasia at the gastroesophageal junction, Barrett’s esophagus, and esophageal adenocarcinoma. Hence, the controversies regarding screening for and surveillance of Barrett’s esophagus and esophageal adenocarcinoma have become more widely relevant to gastroenterologists around the world. Emerging concepts related to esophageal cancer prevention and early detection include the screening for Barrett’s esophagus using wireless videocapsule endoscopy, and chromoendoscopy, enhanced high resolution endoscopy. There is also interest in improving surveillance for esophageal neoplasia using novel imaging techniques, such as high resolution and high magnification endoscopy, narrow‐band imaging, autofluorescence imaging, and endocytoscopy/endomicroscopy. The enhanced detection of Barrett’s esophagus and esophageal neoplasia become even more clinically relevant because of accumulating data on the safety and effectiveness of mucosal ablative techniques (such as photodynamic therapy, argon plasma coagulation, low pressure cryotherapy) and endoscopic mucosal resection. This article summarizes the latest developments related to Barrett’s esophagus that are of interest to endoscopists from the East or West.     

USEFULNESS OF MAGNIFYING ENDOSCOPY IN UPPER GASTROINTESTINAL TRACT: HISTORY AND RECENT STUDIES

Although clinical trials using magnifying optical endoscopy have been reported, magnifying endoscopies have been remarkably developed in the period of electronic endoscopy. Magnifying electronic endoscopies with 80 or 100‐fold magnification are used for routine endoscopic examination of upper gastrointestinal tract in Japan. Magnifying endoscopy is used to visualize the microstructure and microvascular architecture of gastrointestinal surface mucosa. Microsurface structure of the mucosa includes normal structure, changed structure by inflammation and biological response, and tumor‐specific structure. Microvascular architecture includes normal vascular system and tumor microvessels. Magnifying endoscopy is starting to play an important role in diagnosis of any upper gastrointestinal diseases by assessment of magnified observation. Magnifying endoscopy holds a great deal of promise in the near future because magnifying endoscopic observation is approaching optical biopsy.     

Magnifying chromoendoscopy for the detection of premalignant gastrointestinal lesions

The prognosis for patients with malignancies of the gastrointestinal tract is strictly dependent on the early detection of premalignant and malignant lesions. At present, endoscopy can be performed with new, powerful high-resolution or magnifying endoscopes. Comparable to the rapid development in chip technology, the optic features of the newly designed endoscopes offer resolutions that allow new mucosal surface details to be seen. In conjunction with chromoendoscopy, the newly discovered tool of video endoscopy is much easier to use and more impressive than previously used fibreoptic endoscopy. This review summarises the value of magnifying endoscopy in the upper and lower gastrointestinal tract and focuses on gastroesophageal reflux disease and early gastric and colorectal cancer.     

What can we see with the endoscope? Present status and future perspectives

The present status and future perspectives in new technologies of image processing and analysis, infrared ray endoscopy and autofluorescence endoscopy for gastrointestinal cancer are presented in this paper. Spectroscopic measurements using an endoscopic spectroscopic system are useful for distinguishing between benign and malignant gastric mucosal lesions, and the histological classification of early gastric cancer is possible on the basis of the spectroscopic characteristics. It is expected that adaptive hemoglobin index color enhancement would be useful for the qualitative diagnosis of early gastric cancer and for detecting specialized columnar epithelium in Barrett’s esophagus in combination with magnifying endoscopy. Our preliminary experience suggess that magnifying endoscopy with a narrow‐band imaging system could predict the histological characteristics of gastric cancerous lesions with high accuracy. Recent studies revealed that infrared ray electronic endoscopy is very useful for diagnosing the depth of invasion in early gastric cancer. In addition, it is evident that specific antibodies tagged with the indocyanine green derivative can label cancer cells and can generate a fluorescent signal strong enough to detect small cancers using an infrared fluorescence endoscope. The future development and evaluation of autofluorescence endoscopy are discussed, and we propose a modification to the system, including the excitation lights.     

Endoscopic imaging: How far are we from real-time histology?

Currently,in gastrointestinal endoscopy there is increasing interest in high resolution endoscopic technologies that can complement high-definition white light endoscopy by providing real-time subcellular imaging of the epithelial surface.These ’optical biopsy’ technologies offer the potential to improve diagnostic accuracy and yield,while facilitating real-time decision-making.Although many endoscopic techniques have preliminarily shown high accuracy rates,these technologies are still evolving.This review will provide an overview of the most promising high-resolution imaging technologies,including high resolution microendoscopy,optical coherence tomography,endocytoscopy and confocal laser endoscopy.This review will also discuss the application and current limitations of these technologies for the early detection of neoplasia in Barrett’s esophagus,ulcerative colitis and colorectal cancer.     

Magnifying endoscopic observation of superficial esophageal carcinoma

The depth of tumor invasion of esophageal cancers is one of the most important indicators for predicting lymph node metastasis, so much effort has been directed toward improving the diagnosis of tumor invasion, especially in cases of super?cial esophageal cancer. Ultra‐high magnifying endoscopic observation for esophageal cancer was performed using an Olympus Q240Z, which has a 100 × magnifying capacity. We succeeded in observing looped capillary vessels inside the papillae (intrapapillary capillary loop: IPCL). The IPCL inside an m1 cancer showed abnormal changes such as ‘dilation, weaving, changes in caliber, variety of shapes’. Furthermore, we found that super?cial esophageal cancers show characteristic changes according to the depth of invasion. In our investigation, the rate of accurate diagnosis using magnifying endoscopy for super?cial esophageal cancers was 83.1% in cases for which ?ne pictures were obtained. Observations of the microvascular architecture of super?cial esophageal carcinoma using magnifying endoscopy are useful for diagnosing the depth of tumor invasion, especially for super?cial cancers with invasion reaching to the muscularis mucosae (m3) and slightly into the submucosa (sm1) esophageal cancer.     

Early diagnosis of esophageal adenocarcinoma

The incidence of esophageal adenocarcinoma has increased dramatically in developed countries during the past 2 decades, and prognosis remains very poor. Gastroesophageal reflux disease is an important risk factor for this cancer that develops in patients with specialized esophageal metaplasia (Barrett's esophagus). Careful periodic endoscopic examinations, with random biopsy sampling of the entire mucosal surface, are usually recommended for the surveillance of these patients. Several innovative techniques have recently been developed to improve the accuracy of diagnosis of intestinal metaplasia, dysplasia, and early adenocarcinoma in Barrett's esophagus. Some of these techniques (eg, chromoendoscopy, magnifying endoscopy, and light-induced fluorescence endoscopy) are intended to identify suspicious areas of the mucosa not visible during conventional endoscopic examination and to perform targeted biopsies toward these areas to avoid sampling errors. Optical coherence tomography and confocal laser scanning microscopy are other powerful techniques that provide real-time cross-sectional tissue images at a resolution close to that of histology. They allow tissue characterization based solely on optical properties and are likely to replace excisional biopsies. Although promising, none of these techniques is currently recommendable for routine surveillance of patients with Barrett's esophagus. Further evaluation is warranted to define the optimal method and standardize the procedures.     

上消化道肿瘤高危人群放大内镜检查中西部专家意见

食管癌和胃癌是我国中西部地区常见的恶性肿瘤之一,严重威胁中西部地区居民生命健康。近年来,放大内镜技术在早期食管癌和早期胃癌筛查和诊断中已受到广泛使用,国内尤其是中西部众多专家通过多次讨论,并结合上消化道早期癌放大内镜诊治的最新研究进展,制定了更适合我国的上消化道肿瘤高危人群放大内镜检查中西部专家意见,以期进一步提高中西部地区上消化道早期癌的检出率,并推动放大内镜的规范应用。     

Optical biopsy: a new frontier in endoscopic detection and diagnosis.

Endoscopic diagnosis currently relies on the ability of the operator to visualize abnormal patterns in the image created by light reflected from the mucosal surface of the gastrointestinal tract. Advances in fiber optics, light sources, detectors, and molecular biology have led to the development of several novel methods for tissue evaluation in situ. The term "optical biopsy" refers to methods that use the properties of light to enable the operator to make an instant diagnosis at endoscopy, previously possible only by using histological or cytological analysis. Promising imaging techniques include fluorescence endoscopy, optical coherence tomography, confocal microendoscopy, and molecular imaging. Point detection schemes under development include light scattering and Raman spectroscopy. Such advanced diagnostic methods go beyond standard endoscopic techniques by offering improved image resolution, contrast, and tissue penetration and providing biochemical and molecular information about mucosal disease. This review describes the basic biophysics of light-tissue interactions, assesses the strengths and weaknesses of each method, and examines clinical and preclinical evidence for each approach.     

ANGIOGENESIS IN SUPERFICIAL ESOPHAGEAL SQUAMOUS CELL CARCINOMA: MAGNIFYING ENDOSCOPIC OBSERVATION AND MOLECULAR ANALYSIS

Observations of esophageal squamous cell carcinoma using magnifying endoscopy have now been carried out extensively and, as a result, it has become clear that the morphology of the microvessels evident at the tumor surface reflects the depth of tumor invasion. In M1 and M2 cancer, the surface microvasculature reveals dilation and elongation of the intrapapillary capillary loops (IPCL). However, at this stage, some immature capillaries resembling IPCL also arise inside the tumor and, therefore, the view of the microvasculature should be described as one showing ‘intermixing of modified IPCL and IPCL‐like immature capillaries (IPCL‐like abnormal capillary)’. As cancer invades into the muscularis mucosa (M3 or deeper), an obviously dilated and irregularly branched tumor‐specific vasculature, more accurately described as ‘neovasculature’, can be observed. From our magnifying endoscopy observations and studies of the molecular profile of early esophageal cancer, we conclude that two major angiogenic steps exist in precancerous and M3 lesions in the early phase of cancer progression. In addition, it is now possible to study cell morphology using an endocytoscope with a much higher magnification (×400–×1000) than magnifying endoscopes currently on the market. The histology revealed in this way may reduce the need for conventional biopsy histology in the future.     

Magnifying endoscopy with narrow band imaging for predicting the invasion depth of superficial esophageal squamous cell carcinoma

The invasion depth of superficial esophageal squamous cell carcinoma is important in determining therapeutic strategy. The aim of this study was to prospectively investigate the clinical utility of magnifying endoscopy with narrow band imaging compared with that of non‐magnifying high‐resolution endoscopy or high‐frequency endoscopic ultrasonography in predicting the depth of superficial esophageal squamous cell carcinoma. The techniques were carried out in 72 patients with 101 superficial esophageal squamous cell carcinomas, which were then resected by either endoscopic mucosal resection or esophagectomy. The histological invasion depth was divided into two: mucosal or submucosal carcinoma. We investigated the relationship between endoscopic staging and histology of tumor depth. Non‐magnifying high‐resolution endoscopy, magnifying endoscopy with narrow band imaging, and high‐frequency endoscopic ultrasonography had overestimation/underestimation rates of 7/5, 4/4 and 8/3%, respectively. The sensitivity rates for the three techniques were 72, 78, and 83%, respectively, and the specificity rates were 92, 95, and 89%, respectively. There were no statistically significant differences among the three endoscopic techniques. Clinical utility of magnifying endoscopy with narrow band imaging does not seem to be significantly different from that of non‐magnifying high‐resolution endoscopy or high‐frequency endoscopic ultrasonography in predicting the depth of superficial esophageal squamous cell carcinoma. Magnifying endoscopy with narrow band imaging may have potential to reduce overestimation risks of non‐magnifying high‐resolution endoscopy or high‐frequency endoscopic ultrasonography.     

Barrett''s esophagus: current and future role of endosonography and optical coherence tomography

This paper reviews the role of endosonography and optical coherence tomography (OCT) for imaging of Barrett's esophagus (BE). The routine use of endoscopic ultrasound (EUS) to screen patients with BE is neither justified nor cost effective. EUS does appear to have a role in patients who have BE and high-grade dysplasia or intramucosal carcinoma, in whom a non-operative therapy is being contemplated. For patients with a diagnosis of esophageal cancer with or without BE, EUS is superior to computed tomography or magnetic resonance imaging for assessing esophageal wall penetration and for detecting regional lymph node involvement. In its current state, OCT is not yet ready for application in clinical practice. However, given its superior resolution compared with other modalities such as EUS, OCT has great potential as a powerful adjunct to standard endoscopy in surveillance of BE and may enhance the ability of endoscopists to detect high-grade dysplasia at an early stage. With further technical refinement, this technique may become a mainstay in the surveillance of BE and other premalignant conditions of the gastrointestinal tract.     

Does Risk of Progression from Barrett’s Esophagus to Esophageal Adenocarcinoma Change Based on the Number of Non-dysplastic Endoscopies?

Background

There is a large Barrett’s esophagus patient population undergoing endoscopic surveillance. Methods to stratify patients into higher and lower risk groups may enable more varied surveillance intervals for patients with non-dysplastic Barrett’s esophagus that could optimize use of endoscopy resources.

Objective

We aimed to assess whether risk of progression to esophageal adenocarcinoma differed in patients with multiple endoscopic biopsies negative for dysplasia.

Methods

We conducted a retrospective cohort study among individuals from the population-based Northern Ireland Barrett’s register with a histologically confirmed diagnosis of non-dysplastic Barrett’s esophagus (with intestinal metaplasia) between 1993 and 2010, who had at least one endoscopic biopsy conducted at least 12 months after diagnosis. We used Poisson regression to estimate incidence rate ratios (IRR) and 95% confidence intervals (CI) for the association between number of successive endoscopies showing non-dysplastic Barrett’s esophagus and risk of esophageal adenocarcinoma alone, and combined with high-grade dysplasia, at the next endoscopy.

Results

We identified 1761 individuals who met our eligibility criteria. Subsequent risk of progression to esophageal adenocarcinoma was lower at the next endoscopy following two endoscopies showing non-dysplastic Barrett’s esophagus (IRR 0.26, 95% CI 0.10–0.66) than following one endoscopy showing non-dysplastic Barrett’s esophagus. Similar findings were apparent for risk of progression to esophageal adenocarcinoma or high-grade dysplasia (IRR 0.41, 95% CI 0.22–0.79).

Conclusion

The lower risk of malignant progression in individuals with persistent non-dysplastic Barrett’s esophagus over two consecutive endoscopic biopsies but not for longer term persistence does not support hypotheses of persistence being an indicator of less biologically aggressive lesions. Instead, the initial difference may be attributable to post-endoscopy cancers and support the necessity of adhering to robust quality standards for endoscopic procedures.

     

ENDOCYTOSCOPY FOR ESOPHAGEAL CANCER EX VIVO

Endocytoscopy is a novel magnifying endoscopy method that provides images of living cells. We performed endocytoscopy on the fresh esophagus with 0.5% methylene blue. The images of normal esophagus and esophageal cancer by the endocytoscopy wers consistent with the horizontal histological pictures. This new modality will show the optical biopsy under ongoing endoscopy.     

放大结合窄带成像在上消化道内镜检查中指导靶向活检的价值

目的探讨放大内镜（magnifying endoscopy,ME）结合窄带成像（narrow—band imaging,NBI）在上消化道胃镜检查中指导靶向活检的价值。方法筛选普通白光胃镜检查活检提示存在低级别上皮内瘤变的患者或直径大于2．0cm的胃溃疡患者,共200例,随机分成2组,2—4周复查胃镜。普通白光胃镜组：普通白光胃镜观察后局部活检。ME—NBI组：根据放大胃镜下表现,在病变最严重部位靶向活检。分析普通胃镜活检结果与ME—NBI靶向活检结果与最终病理诊断结果的关系。结果200例患者中,3例患者失访,共完成197例。普通白光胃镜组100例,其中食管病变23例,胃病变77例。ME—NBI组97例,其中食管病变19例,胃病变78例。ME-NBI组平均每例活检数（2．95块）与普通胃镜组（4．56块）相比差异具有统计学意义（P〈0．001）。与最终病理结果符合率：ME—NBI90．7％（88／97）,普通胃镜71．0％（71／100）。两组间差异具有统计学意义（P〈0．01）。结论ME—NBI技术操作简便,可清晰观察病灶微细结构,有助于提高早期上消化道肿瘤的靶向活检准确率,在常规胃镜检查中发现可疑病灶后,可使用ME．NBI观察,指导靶向活检。