The Study Of Microvascular Architecture Alterations In The Diagnosis Of Gastroesophageal Early Cancer And Its Relation With Angiogenesis By Confocal Laser Endomicroscopy

Background:Esophagogastric cancer is the most frequent in China,and the prognosis is poor. Most cases of esophagogastric cancer are detected at the advanced stage,and this explains the overall poor prognosis.Early diagnosis improves survival.The screening campaign conducted in Japan increased the proportion of early-stage tumors to around 40%,and recent figures show that the five-year survival increased from around 20% in 1965 to over 40%in 1992.In the esophagus,the risk of positive lymph nodes is near to zero in intramucosal cancer,while submucosal cancer is regarded as an advanced stage,with a 35%risk of positive nodes.Early detection is still regarded as the best option for improving the prognosis in patients with esophagogastric cancer and endoscopy is the commonest technique of earlier diagnosis.However,traditional endoscopy has many limitations to diagnosis early cancer.Within the past two decades,advances in biomedical optics have been applied to overcome the limitations of conventional endoscopy for detecting dysplasia and early cancer in esophagus and stomach.It has been suggested that chromoendoscopy and magnifying endoscopy provide numerous details of the esophageal surface.In recent years,much emphasis has been placed on developing new gastrointestinal endoscopy technologies or techniques in order to provide a precise and even a "real time" endoscopic diagnosis.Endocytoscopy and confocal endomicroscopy stand at the forefront of the novel endoscopic techniques.Endocytoscopy with 400～1100×magnification has been integrated into regular endoscopes and the shape of epithelial cells,nucleus and cytoplasm could be identified clearly.In addition to the minute structure of the mucosa,the microvascular pattern in the cancerous lesion is very important for determining a tumor's malignant potential. Neovascularization and changed capillary vessel density in neoplasia make the assessment of capillary pattern critical in the diagnosis of dysplasia and early cancer. Traditional method to determine microvascular density(MVD)is immunostaining with CD34,CD31,factorⅧon paraffin or freezing specimen.However,the microvascular characteristic such shape and caliber in specimen can't reflect the condition in vivo due to dehydration in treating process.New type endoscopy that could observe microvasculature during endoscpy is earnest expectation of endoscopists.Though magnification endoscopy and narrow band imaging(NBI)can observe mucosal vessels reported by many studies,the resolution is limitation.Confocal laser endomicroscopy(CLE)is a newly introduced endoscopic tool that allows the observation of not only living cells and tissue but also the vascular networks of the mucosal layer in the gastrointestinal tract during ongoing endoscopy. The newly type endoscopy enables the observation of the images magnified up to 1000 folds,which could has a visualization of the mucosal microvascular architecture in units as small as the capillary in the mucosal layer.The optical slice thickness is 7μm with a lateral resolution of 0.7μm.The field of view is 475×475μm.The range of the z-axis was 0-250μm below the surface layer.Therefore,CLE provides images in a horizontal axis,whereas conventional histology allows differentiation of the mucosal and submucosal layers on a vertical axis.The clinical application of CLE need badly to identify the confocal images,analysis the difference between normal and cancerous mucosa on confocal images,construct various diagnosis parameters in order to assess its clinical application value.The present research is to describe the characteristics of normal esophagogastric microvascular in confocal images,evaluate the feasibility of in vivo real-time simulation histopathology by confocal endomicroscopy.And we are going to analyze the distinct characteristics of microvasculature between normal and cancerous mucosa on confocal images,determine various diagnosis parameters and assess the clinical application value of CLE.Besides,we will analyze the relation between confocal images and corresponding histoimmunostaining findings,assess the the feasibility of in vivo observation of tumor angiogenesis.Objectives:1.The first experiment is to assesse the feasibility of in vivo detecting superficial vascular architecture by confocal endomicroscopy in normal esophagogastric mucosa and defined the characteristics of the vasculature,and evaluated CLE as a tool for conducting virtual biopsies of the gastroesophageal mucosa by comparing confocal images with biopsy samples.2.The second experiment is to define the endomicroscopic characteristics of cells and intrapapillary capillary loops(IPCLs)on the normal and malignant esophageal mucosa and assess the potential application of CLE to detect superficial esophageal squamous-cell carcinoma(SESC).3.The distinct characteristics of microvasculature between normal and cancerous gastric mucosa on confocal images was drawn out,then various diagnosis parameters were determined and the capability of CLE to diagnose gastric neoplasia was evaluated.4.We investigated the relation between the caliber and number of confocal images and the microvessel density marked with CD34,CD 105 and VEGF expression of the corresponding biopsy tissue in the normal gastric mucosa-LGEN-HGEN-EGC sequence.We assessed the possibility of observing angiogenesis in vivo by CLE and investigated whether an angiogenic switch would take place during the malignant transformation.Methods1.We recruited 32 patients without any symptoms who underwent endoscopy for routine physical examination or follow-up after polypectomy in the study between July 2006 and June 2007.The esophagus and stomach including gastroesophageal junction were examined by confocal endomicroscopy using an intravenous injection of fluorescein and biopsy was performed.The confocal images with microvasculature were collected and the characteristics were analyzed.The confocal images were compared with conventional histopathological results from biopsied samples.2.Consecutive patients with superficial esophageal carcinoma diagnosed at conventional endoscopy and confirmed by histopathology between July 2006 and June 2007 in Qilu Hospital were recruited.Confocal endoscopic images were compared with corresponding histology.The characteristic pattern of cells and IPCLs in confocal endoscopic images from malignant and normal mucosa was compared. The quality of images and interobserver variations of the involved endoscopists to predict histology were evaluated.3.We recruited 61 patients with early gastric carcinoma or dysplasia diagnosed by conventional endoscopy and confirmed by histology in the third study.Two of neoplastic areas and two of non-neoplastic mucosa of each patient were examined by confocal laser endomicroscopy.Confocal images were charactered according to microvascular architecture pattern,caliber,number,area and vessel leakage.The vascular pattern was correlated with the histological findings of targeted biopsy specimen.Interobserver variations of the involved endoscopists to predict histology were evaluated.4.We recruited 23 sites with differentiated early gastric cancer(DEGC),16 sites with undifferentiated early gastric cancer(UEGC),20 sites with high grade epithelial neoplasia(HGEN),20sites with low grade epithelial neoplasia(LGEN)and 10 sites with normal gastric mucosa(NG)of previous study in the present study.The confocal images were collected.The corresponding tissue was immunostained with CD34,CD 105 and VEGF.The expression of CD34,CD105 and VEGF were compared with the caliber and number in confocal images.Results1.In the first study,the GI mucosa turned yellow 10 to 20 seconds after intravenous injection of fluorescein.The mean duration of the endomicroscopic examination in all thirty-two patients was 25 minutes.Among the sites of esophagus, 11 of 32 were observed paliform vascular net which have different size caliber and natural shape.Normal squamous epithelium had a regular intraepithelial papillary capillary loops(IPCLs)directed toward the luminal surface.Honeycomb-like and coil-shaped regular microvascular architecture surrounding gastric pits were visible in normal gastric body and antrum respectively.All of the CLE images observed were concordant histopathologically with the biopsy results.2.In the second study,overall 129 sites(69 SESC,60 controls)from 38 patients (18 SESC,20 controls)were examined by CLE.The confocal images corresponded to the H&E staining from the same sites.Significantly higher proportion of patients with SESC had squamous epithelial cells with irregularly arrangement,increased diameter of IPCLs,and abnormal shape IPCLs compared to controls.In the SESC group, massive IPCLs with tortuous vessels,long branching IPCLs were seen more frequently.34.0%of all the images were graded as good quality.The interobserver agreement for the prediction of cancerous mucosa was graded as substantial.3.In the third study,there were 239 areas detected of microvascular architecture by confocal endomicroscopy.TypeⅠvascular pattern are present at 23 area(69.6%) of DEGC;typeⅡpattern are present at 16 area(75.0%)of UEGC;80.6%of HGEN are present with typeⅢvascular pattern;34.0%and 48.9%of LGEN are present with typeⅣandⅤ;typeⅣpattern are present at 95.1%of NGB areas;typeⅤpattern are present at 98.4%of NGA areas.The caliber of DEGC,UEGC and HGEN were higher than LGEN and NG.The number of DEGC and HGEN were more than NG.The area of DEGC was more than UEGC,however,it wasn't significant deviation with NG.Vessel leakage are present more frequently in EGC than in HGEN, NGB and NGA.39.3%of all the images were graded as good quality.Theκvalue for the interobserver agreement was graded as good.4.The count of MVD-CD34 was increased in DEGC and HGEN compared with UEGC,LGEN and NG.The count of MVD-CD105 of DEGC,UEGC and HGEN was higher NG.VEGF expression in DEGC,UEGC and HGEN was increased.The number in confocal images was correlated well with MVD-CD34,and it was correlated with MVD-CD105.The caliber in confocal images in sites of positive expression of VEGF was significantly increased than that of negative expression.Conclusions:1.Confocal laser endomicroscopy system could yield clear images of superficial microvascular network in normal gastroesophageal mucosal layer.CLE provides endoscopists with a valuable new diagnostic tool for observing tissue in situ at the histopathological level.2.The ability of CLE to differentiate between normal and cancerous squamors epithelium gives it potential value for early detection of esophageal carcinoma.3.The detailed and characteristic changes of microvascular architecture of neoplastic mucosa and normal mucosa in gastric tissue could be identified using the confocal endomicroscopy,by which the histological characteristics of gastric neoplasia could be predicted with a high accuracy.It seems to represent a very useful new technology for diagnosing gastric neoplasia.4.In the vascular characteristics of confocal images,vascular pattern,caliber, number are valuable diagnostic parameters,by which we can predict early gastric cancer and SESC.5.The confocal images were correlated well with corresponding tissue immunostaining with CD34,CD105 and VEGF.It is feasible of observing angiogenesis in vivo by CLE and an angiogenic switch may take place during the stage of HGEN.Significance:1.The study described the confocal microvascular characteristics of normal esophageal epithelium,normal gastric mucosa,superficial esophageal squamous-cell carcinoma and early gastric cancer.2.The study analysed systematically the difference between normal and malignant mucosa,determined the diagnosed parameter of microvasculature such as pattern,caliber,number,area and leakage,and evaluated the clinical value of the parameter to diagnose early esophagogastric cancer.3.We designed confocal images analysis software to measure caliber and area of confocal images,which improved the practicability and convenience of CLE.4.The proposition was drawn out that an angiogenic switch may take place during the stage of HGEN in gastric cancer carcinogenesis.5.The study evaluated the feasibility of observing angiogenesis in vivo by CLE, which provided reliable evidence of clinical anti-angiogenic therapy.