Staining and magnifying the mucosa: are biopsies no longer required?

William Shakespeare asked in his Sonnet 101: “wilt though not haply say, ‘Truth needs no colour, with his colour fixed; Beauty no pencil, beauty’s truth to lay; But best is best, if never intermixed’?”

Since the introduction of the first semiflexible endoscopes in the 1930’s and 40’s, and the development of the first flexible fiberoptic endoscope by Basil I. Hirschowitz in 1957, endoscopic technology has improved dramatically [1]. Besides the development of more flexible devices, endoscopic imaging has seen many improvements. Fiberoptics have been replaced by charge coupled devices (CCDs) allowing standard-resolution, high-resolution, and more recently even high-definition white-light imaging. Most recently, complementary metal-oxide semiconductor (CMOS) optics have been introduced, enabling high-definition imaging with significantly less noise in comparison to CCD optics.

Endoscopy was first developed as a tool purely for the detection of lesions within the luminal gastrointestinal tract. Evaluation of lesions was dependent on the instrument used. Whereas endoscopists using fiberoptic scopes focused on describing the visual experience, endoscopists using the gastrocamera obtained dozens of photographs of the stomach lining. The pictures were then developed, scrutinized, and analyzed, and a diagnosis was made. However, a final diagnosis was still always dependent on the final pathological results.

In an effort to increase the in vivo diagnostic accuracy, endoscopists have developed various techniques that allow them to better describe the mucosal changes, such as the water-immersion technique. Flushing water into the luminal gastrointestinal tract meant that “magnification” endoscopy became possible and indeed this has shown its utility in the non-histological diagnosis of celiac disease [2].

Shortly after this, optical magnification endoscopes were introduced, which allowed magnification of up to 150-fold. However, despite the use of optical magnification, tissue contrast remained insufficient to adequately analyze the mucosa. Therefore, dyes were topically applied onto the mucosa in order to improve tissue contrast for high-magnification imaging (“chromoendoscopy”). In 1996, Professor Kudo introduced the outstanding pit pattern classification of colorectal polyps [3]. He showed that by using optical magnification in combination with chromoendoscopy in vivo prediction of colorectal polyp histology became feasible.

Because dye-based chromoendoscopy suffers from potential limitations, including a prolonged procedure time and additional costs for dye-spraying, dye-less chromoendoscopy techniques have now been introduced [4]. Initially based on optical filters that narrowed the red light (“narrow-band imaging”), these newer dye-less chromoendoscopy techniques use digital post-processing to improve tissue contrast. However, and in contrast to optical chromoendoscopy, digital chromoendoscopy techniques do not allow for a detailed analysis of the morphology of the mucosal vascular pattern. As a result, the newest generation endoscopes are now using a combination of optical and digital chromoendoscopy [5].

Various studies have shown the effectiveness of dye-less chromoendoscopy, with or without optical magnification, for the prediction of pathology. Nevertheless, physical biopsies and subsequent histopathological analysis of the tissue still remain the gold standard for diagnosis. Significant limitations of this approach remain, however, including the prolonged time until the final diagnosis, the small sample size obtained, sampling error, and the induction of submucosal fibrosis after physical biopsies, which may subsequently limit endoscopic therapy, and as a result, optical biopsy techniques have been introduced.

The new techniques include confocal laser endomicroscopy (Pentax, Tokyo, Japan and Mauna Kea Technologies, Paris, France), endocytoscopy (Olympus, Tokyo, Japan), and the WavSTAT-system (SpectraScience, San Diego, California, USA). The last of these is based on the principle of spectroscopy and only limited data are as yet available on the effectiveness of the device. Endomicroscopy is based on tissue illumination after the application of fluorescent agents and enables magnification of up to 1000-fold. Images are displayed in black and white at up to 12 frames per second. Finally, endocytoscopy allows in vivo imaging at a magnification of up to 1390-fold after the application of dyes [6]. The technique is based on the principle of contact light microscopy. The main advantage of endocytoscopy is the observation of cellular detail, including the appearance of the nucleus and assessment of the nucleus-to-cytoplasm ratio, which is essential for the diagnosis of dysplasia, while the main disadvantage is that imaging of only the very superficial mucosal layer is possible, thereby limiting its usefulness for invasive diseases.

In this issue of Endoscopy, Kaise and co-workers report for the first time the value of endocytoscopy using 380-fold magnification and a field of view of 700 × 600 µm in the diagnosis of gastric cancer [7]. Staining was performed using topical application of crystal violet, which stains cell walls, and methylene blue, which stains nuclei. After in vivo imaging, lesions were removed for subsequent histopathological analysis.

Trainees and experts reviewed 100 endocytoscopic images, including non-neoplastic lesions (n = 46), low-grade adenomas (n = 10), high-grade adenomas (n = 3), and adenocarcinomas (n = 41), after first viewing a training set of images to standardize image interpretation. Criteria for cancer diagnosis were defined as: (i) lumen absence, (ii) lumen fusion, and (iii) irregular nuclei. The authors calculated a sensitivity, specificity, and accuracy of 78 %, 93 %, and 87 %, respectively, for prediction of high-grade neoplasia. The positive predictive value and negative predictive value (NPV) were calculated as 85 % and 87 %, respectively. Interestingly, image interpretation was not dependent on endoscopic expertise, while interobserver agreement was good with a kappa value of 0.7.

The authors have to be congratulated for this study, which for the first time shows the potential of endocytoscopy for the diagnosis of high-grade neoplasia within the stomach. Recent data have already shown the benefit of endocytoscopy for the diagnosis of colorectal polyps and inflammatory bowel disease [8] [9]. Thus, their study is a valuable addition to the literature on advanced imaging.

One potential drawback of the study is the ex vivo evaluation of the images. In addition, the endoscopists had no information from the white-light or even NBI imaging. Therefore, the study does not reflect a real-life situation and one can only speculate about the additional value of endocytoscopy for the in vivo prediction of gastric pathology. Nevertheless, we speculate that previous information gathered through in vivo analysis of high-definition white-light and dye-less chromoendoscopy (in this scenario NBI) could improve the characterization of lesions and the detection of early cancer. One recent multicenter study evaluated the use of NBI for the diagnosis of gastric precancerous and cancerous lesions, describing accuracies of 83 % and 95 %, respectively, for the diagnosis of histologically normal and dysplastic tissue [10]. This was also confirmed by another study that demonstrated a sensitivity and specificity of 93 % and 95 %, respectively, for the diagnosis of superficial gastric lesions [11].

As the main advantage of optical biopsy techniques is their usage in vivo and endoscopic imaging techniques should work in addition to these, future research should evaluate whether a combined approach of modern imaging techniques will allow adequate in vivo diagnosis of suspicious lesions as well. This is extremely important, especially if it is to be used to guide subsequent endoscopic submucosal dissection (ESD) for sm1 lesions or surgical therapy [12].

Recently, the ASGE PIVI statement proposed that a new technology should provide an NPV > 90 % for adenomatous polyp histology if distal diminutive colorectal polyps are to be left in place without resection [13]. Although this statement is only valid for colorectal lesions, it can be speculated that a similar statement might also be valid for gastric lesions. Notably, the NPV in the study by Kaise et al. did not reach this proposed threshold. In this context, it would be highly interesting to know if the combined approach of (magnifying) NBI and endocytoscopy could yield an increased NPV.

Endocytoscopic imaging is dependent on the application of absorptive dyes after careful removal of mucus and debris from the mucosal surface. This approach often requires repeated staining of the mucosa. Nevertheless, inadequate staining was often a major drawback in previous studies of endocytoscopy. Here, Kaise and co-workers have introduced a novel and smart way to repeatedly stain the mucosa without reinserting the spraying catheter by applying the dyes through the working channel during endocytoscopic imaging at full magnification. This approach may also help to improve endocytoscopic imaging in further studies.

The study by Kaise at al. has shown in an elegant way the potential of endocytoscopy to predict high-grade neoplasia in lesions located in the stomach. However, an approach that avoids standard physical biopsies can not as yet be recommended, especially when the question of neoplasia arises. Or in the words of William Shakespeare, “Truth needs no colour, with his colour fixed; Beauty no pencil, beauty's truth to lay”.

• References

• 1 Mönkemüller K, Neumann H, Fry LC. Endoscopic examination of the small bowel: from standard white light to confocal endomicroscopy. Clin Gastroenterol Hepatol 2009; 7: 11-12
• 2 Cammarota G, Cesaro P, Cazzato A et al. The water immersion technique is easy to learn for routine use during EGD for duodenal villous evaluation: a single-center 2-year experience. J Clin Gastroenterol 2009; 43: 244-248
• 3 Kudo S, Tamura S, Nakajima T et al. Diagnosis of colorectal tumorous lesions by magnifying endoscopy. Gastrointest Endosc 1996; 44: 8-14
• 4 Mönkemüller K, Zimmermann L. An advanced chromocolonoscopic picture is worth a thousand words, but is it worth the effort?. Am J Gastroenterol 2010; 105: 1308-1310
• 5 Neumann H, Fujishiro M, Wilcox CM et al. Present and future perspectives of virtual chromoendoscopy with i-scan and optical enhancement technology. Dig Endosc 2014; 26: 43-51
• 6 Neumann H, Fuchs FS, Vieth M et al. Review article: in vivo imaging by endocytoscopy. Aliment Pharmacol Ther 2011; 33: 1183-1193
• 7 Kaise M, Kimura R, Nomura K et al. Accuracy and concordance of endocytoscopic atypia for the diagnosis of gastric cancer. Endoscopy 2014; 46: 827-832
• 8 Kudo SE, Wakamura K, Ikehara N et al. Diagnosis of colorectal lesions with a novel endocytoscopic classification – a pilot study. Endoscopy 2011; 43: 869-875
• 9 Neumann H, Vieth M, Neurath MF et al. Endocytoscopy allows accurate in vivo differentiation of mucosal inflammatory cells in IBD: a pilot study. Inflamm Bowel Dis 2013; 19: 356-362
• 10 Pimentel-Nunes P, Dinis-Ribeiro M, Soares JB et al. A multicenter validation of an endoscopic classification with narrow band imaging for gastric precancerous and cancerous lesions. Endoscopy 2012; 44: 236-246
• 11 Kato M, Kaise M, Yonezawa J et al. Magnifying endoscopy with narrow-band imaging achieves superior accuracy in the differential diagnosis of superficial gastric lesions identified with white-light endoscopy: a prospective study. Gastrointest Endosc 2010; 72: 523-529
• 12 Kobara H, Mori H, Fujihara S et al. Prediction of invasion depth for submucosal differentiated gastric cancer by magnifying endoscopy with narrow-band imaging. Oncol Rep 2012; 28: 841-847
• 13 Rex DK, Kahi C, O'Brien M et al. The American Society for Gastrointestinal Endoscopy PIVI (Preservation and Incorporation of Valuable Endoscopic Innovations) on real-time endoscopic assessment of the histology of diminutive colorectal polyps. Gastrointest Endosc 2011; 73: 419-422