Confocal Laser Endomicroscopy For Diagnosis Gastric Intestinal Metaplasia In Vivo

Background and aimsGastric cancer is the second leading cause of cancer related mortality worldwide. Gastric intestinal metaplasia(GIM)is a risk factor that leads to the development of intestinal-type gastric cancer and is generally regarded as a precancerous condition.IfGIM was identified under endoscope earlier,effective strategies could be developed to detect the early,curable phase of gastric cancer and prevent its progression.GIM is multifocal and is mostly indistinguishable by the conventional endoscopy or even by other new endoscopic techniques.Conventional endoscopic identification of intestinal metaplasia has a high rate of interobserver variability and correlates poorly with the histological finding.None of them can distinguish the structure of individual cells,or allow the endoscopic criteria of GIM to be defined. Histologic analysis of biopsy material remains the gold standard for the final diagnosis of GIM.Recently,confocal laser endomicroscopy(CLE)has been developed which is integration of a confocal laser microscope in the distal tip of a conventional videoendoscope.The components enable confocal microscopy in addition to standard videoendoscopy.The new device can provide real-time,high magnification, cross-sectional images of the gastrointestinal epithelium during routine endoscopy without the need for biopsy and thus histopathology has been termed optical biopsy. Compared with other new optics techniques,the greatest advantage of the CLE is that it can enables surface and subsurface imaging of living cells in the mucosa during ongoing colonoscopy.The confocal images that approximately 1000-fold magnification readily permits single cells in the gastrointestinal tract to be resolved.The aim of this study were to determine if CLE could identify cells and subcellular structures of normal and intestinal metaplasia mucosa,definite the confocal criteria of GIM,definite the criteria of its grading and subtype,and evaluate the efficacy of CLE for the extent and topographic patterns of GIM assessment in vivo.Methods1 Identification of cells and subcellular structures in gastric intestinal metaplasia by confocal laser endomicroscopyPatients with known GIM underwent CLE(Pentax EC-3870K;Pentax,Tokyo, Japan).Fluorescein sodium and acriflavine hydrochloride was used as contrast agent. Esophagus,stomach and duodenum were examined with the CLE system.In normal esophagus,squamous epithelial cell showed rhombus single cells at high resolution with clear visible borders.The nucleus can be stained clearly with acriflavine hydrochloride.Furthermore,squamous epithelial cells were cultured and observed with CLE in vitro for identification.Gastric type columnar epithelial cells and mucin-containing goblet cells can easily recognized in stomach under CLE images.In some areas,a more slender,and brighter than columnar epithelial cells of normal gastric mucosa can be seen with a clear dark line at the surface of the epithelium. These cells and structures also are seen in duodenum.The histologic specimens from each site were compared with the targeted confocal images.All of the biopsy specimens were sectioned vertically and transversely to facilitate the comparison between histology and confocal images.All the biopsy specimens were stained with hematoxylin and eosin.Biopsy specimens diagnosed as GIM and duodenum were further stained by AB-PAS mucin staining,HID-AB mucin staining and CD10 immunohistochemistry.These slender and brighter cells and the clear dark line at the surface of the epithelium were identified with scanning electron microscope(SEM) and transmission electron microscope(TEM). 2 Definition and evaluation of gastric intestinal metaplasia with confocal laser endomicroscopy in VivoIn first phase,28 patients with known GIM underwent CLE,and CLE criteria for diagnosis of GIM were developed.In addition,53 consecutive patients with known or suspected GIM were prospectively evaluated in second phase.Standard and abnormal appearance areas were examined with the CLE system.Fluorescein was used as contrast agent.Afterwards,a targeted biopsy was done at the same sites.The results of histoathological biopsy specimens taken from the corresponding sites of gastric mucosa under CLE were regarded as gold standard.3 Diagnostic value of confocal laser endomicroscopy for the prediction of the subtype and grading of gastric intestinal metaplasiaIn first phase,28 patients with known GIM underwent CLE,and CLE criteria for classifying and grading GIM were developed.In addition,53 consecutive patients with known or suspected GIM were prospectively evaluated in second phase. Standard and abnormal appearance areas were examined with the CLE system. Fluorescein was used as contrast agent.Afterwards,a targeted biopsy was done at the same sites.The results of histoathological biopsy specimens taken from the corresponding sites of gastric mucosa under CLE were regarded as gold standard.The expression of three gastric carcinoma related gene were detected in different subtype and severity GIM.4 Diagnostic value of confocal laser endomicroscopy for the prediction of the extent and topographic patterns of gastric intestinal metaplasiaSeventy patients with known GIM underwent CLE.Fluorescein was used as contrast agent.Endoscopic gastric biopsy specimens were obtained using a jumbo forceps from the 11 gastric sites.Five specimens were from the antrum,six from the corpus and one from the incisura angularis.The presence of GIM was made immediately by the endoscopist at the time of the procedure.The extent of GIM was categoried focal,multifocal and extensive GIM.Four topographical patterns of intestinalization emerged:"Focal,","Antrum-predominant,Magenstraβe" and "Diffuse,".The histological evaluation remains the gold standard for the final diagnosis of intestinal metaplasia.Results1 All of 28 patients under went CLE with known GIM.A total of 5750 CLE images were obtained from 124 areas under CLE images.The confocal images showed the normal squamous epithelial cells their nucleus on the surface of the esophagus at high resolution in vivo.These patterns can be directly compared with cultured cells in vitro and H&E-stained sections of biopsy specimens cut parallel to the tissue surface.At the surface of the stomach,epithelium a typical cobblestone structure.The mucin-containing goblet cells showed very dark and big within the columnar-lined epithelium.Goblet cells had specific appearance and easily recognized.The more slender and brighter than columnar epithelial cells of normal gastric mucosa were identified as absorptive columnar epithelial cells.These cells were colorless by AB/PAS and HID/AB mucin staining,difference from goblet cells (blue)and gastric epithilum(purple).The clear dark line at the surface of the epithelium were identified as brush border by CD 10 immunohistochemistry,SEM and TEM.2 GIM was identified if any of the following three features were present in an image field:goblet cells,columnar absorptive cells and brush border,and villiform foveolar epithelium.In a prospective study,a total 267 sites from 53 patients were obtained.160 from 36 patients were diagnosed histopathologically as GIM.The sensitivities of conventional endoscopy and CLE for GIM were 36.88%vs.98.13%, the specificities were 91.59%vs.95.33%,the positive predictive value were 86.76% vs.96.91%,and the negative predictive value were 49.25%vs.97.14%,respectively. The kappa value for the correlation with histological findings was 0.25 for conventional endoscopy vs.0.94 for CLE.3 In the CLE images,GIM was classified as complete or incomplete,according to the shape of goblet cells,the presence of absorptive cells or brush border,and the architecture of vessels and crypts.In a prospective study,a total 267 sites from 53 patients were obtained.Among the 98 sites with complete GIM according to CLE, this was confirmed in 83 by histopathology.The sensitivity,specificity,positive predictive value and negative predictive value of CLE for the diagnosis of complete GIM were 68.03%,89.66%,84.69%and 76.92%,respectively.Among the 64 sites with incomplete GIM according to CLE,this was confirmed histologically in 26.The sensitivity,specificity,positive predictive value and negative predictive value of CLE for the diagnosis of incomplete GIM were 68.42%,83.41%,40.63%and 94.09%, respectively.The kappa score for the agreement between CLE and histopathology was 0.67.Among 146 GIM positive areas,88 were identified as mild GIM by CLE,in which 74 confirmed by histopathology.Thirty-thee areas were diagnosed as moderate GIM by CLE,25 of them were confirmed by histopathology.Twenty-eight areas were diagnosed as marked GIM by CLE,20 of them were confirmed by histopathology. The sensitivity and specificity of CLE were 90.2%and 78.1%for the diagnosis of mild GIM,69.4%and 92.2%for morderate GIM,71.4%and 95.8%for marked GIM,respectively.The kappa coefficient of CLE criteria and the histopathological grading for mild,moderate and marked IM were 0.69,0.64 and 0.70,respectively. There were differences for the expression of CDX2,Ki67 and APC among different subtype and severity GIM.4 A total of 70 gastric carcinoma and chronic atrophy gastritis patients with GIM were recruited.47.1%of them were focal GIM,41.4%were multifocal GIM and 11.4%were extensive GIM.The extent of GIM is associated with the gastric cancer and associated with lesion precancerous.Multifocal GIM and ententive GIM was significantly associated with the presence of cancer and gastric atrophy.Extensive GIM was significantly associated with the presence of cancer and dysplasia.Of the entire study population of 70 subjects,37.1%of them were focal pattern,21.4% were antrum-predominant pattern,31.4%of them were Magenstraβe pattern,10.0% of them were diffuse pattern.The topographic patterns of GIM is associated with gastric cancer and lesion precancerous.MagenstraBe and diffuse topographic patterns was significantly associated with the presence of cancer and dysplasia.Conclusion:Confocal laser endomicroscopy is a newly developed diagnostic tool and may offer an instant and reliable diagnostic tool for in vivo histology.CLE can identify cells and subcellular structures in normal gastric mucosa and GIM at high resolution in vivo,enable classification,grading,extent and topographic patterns of GIM with high accuracy during ongoing endoscopy.SignificanceConfocal laser endomicroscopy is a newly developed diagnostic tool and may offer an instant and reliable diagnostic tool for in vivo histology.CLE can diagnose GIM with high accuracy during ongoing endoscopy,as well as its grading and subtype. The extent and topographic patterns of GIM also be evaluated.GIM is a risk factor that leads to the development of intestinal-type gastric cancer and is generally regarded as a precancerous condition.Most GIMs are only "precancerous conditions" rather than "precancerous lesions".This present study will help determine the role of CLE in screening and surveillance of premalignant conditions.It could serve as a reasonably good predictor of cancer risk and provide appropriate follow-up in an individual patient.Endoscopic confocal imaging systems are revolutionary instruments in the emerging realm of optical biopsy techniques.The new detailed images seen with CLE unequivocally are the beginning of a new era.It is tempting to speculate that CLE will play an important diagnostic role in the future during gastrointestinal endoscopy.