Diagnosis Of Gastric Cancer And Precancerosis In Vivo And Molecular Imagine Of Gastric Cancer By Confocal Laser Endomicroscopy

Background and aims:There is a high incidence of gastric cancer and precancerous lesions in China.Gastrointestinal endoscopy is an effective method to detect the early gastric cancer and precancerosis.Morphological imaging and molecular imaging of confocal laser endomicroscopy(CLE)can play an important role in gastric cancer and precancerous lesions diagnosis.However,there are few studies of the clinical application of CLE.The aims of the study are to find out the capabilities of probe-based CLE(pCLE)in diagnosing gastric cancer and precancerosis in vivo and molecular imaging of gastric cancer.Methods:1.Diagnosis of gastric cancer and precancerosis in vivo by CLE Patients'information was collected according to predefined inclusion and exclusion criteria from December 2013 to November 2014 in the first affiliated hospital of Zhejiang university medical college.Two doctors who had an experience of reading pCLE images over three years read pCLE images of each patient retrospectively and made a diagnosis according to the criteria as followed.The pCLE diagnostic criteria of atrophic gastritis(AG)was decreased gastric pits and dilated opening of the gastric pits.The pCLE diagnostic criteria of gastric intestinal metaplasia(GIM)was goblet cell or columnar absorptive cell or striated border or villiform foveolar epithelium.The pCLE diagnostic criteria of low-grade intraepithelial neoplasia(LGIN)was mild to moderate irregular pit with variable width of the epithelial line and irregular capillary with heterogenous leakage or dilated caliber.The pCLE diagnostic criteria of high-grade intraepithelial neoplasia(HGIN)was prominent distorted pit with irregular epithelial lining and irregular capillary with heterogenous leakage or dilated caliber.The gold standard was the pathologic diagnosis.Sensitivity,specificity,positive predictive value,negative predictive value,positive likelihood ratio,negative likelihood ratio,Youden index and corresponding 95%confident interval(CI),coincidence rate and ? value were calculated.Besides,the pCLE diagnostic criteria of compete GIM was that goblet cells were interspersed between nonsecretory absorptive cells with well delineated striated border.The pCLE diagnostic criteria of incompete GIM was that goblet cells were scattered among gastric type cells or tortuous and branched gastric pits lined by tall columnar cells without striated border.Consistency of pCLE diagnosis and Hematoxylin and Eosin(HE)staining diagnosis was analyzed.2.Molecular imaging of gastric cancer by CLE This part was divided into two steps,including gastric cancer cell lines characterization and experiments of tumor-xenograft nude mice.HER2 expression levers of human gastric cancer cell lines MGC-803 and NCI-N87 were tested by western blot firstly.Then,combining capacity of Alexa Fluor labeling antibody against HER2(AF-anti-HER2)to HER2 over expression cell line was assessed through flow cytometry and cell immunofluorescence experiment.Xenografts were induced by subcutaneous injection of MGC-803 and NCI-N87 cells into the right underarm of nude mice.Macroscopic fluorescence detection in vivo and microscopic fluorescence detection by pCLE were accomplished afterwards.Two experienced doctors read the pCLE images and graded fluorescence into-,+,2+,3+,4+.HE staining and immunohistochemistry of xenograft and liver were finished to confirm the HER2 expression in xenograft and liver.Results:1.Diagnosis of gastric cancer and precancerosis in vivo by CLE 55 patients were included in this study,and 80 gastric lesions were analyzed.Sensitivity,specificity,coincidence rate and ? value of AG diagnosis by pCLE were 81.25%(95%CI,53.69%-95.03%),98.44%(95%CI,90.46%-99.92%),95.00%and 0.84.Sensitivity,specificity,coincidence rate and ? value of GIM diagnosis by pCLE were 93.22%(95%CI,82.73%-97.81%),90.48%(95%CI,68.17%-98.33%),92.50%and 0.81.Coincidence rate and? value of complete GIM diagnosis by pCLE were 0.75 and 0.49.Coincidence rate and? value of incomplete GIM diagnosis by pCLE were 0.79 and 0.48.Sensitivity,specificity,coincidence rate and ? value of LGIN diagnosis by pCLE were 82.35%(95%CI,55.80%-95.33%),88.89%(95%CI,77.84%-95.04%),87.50%and 0.66.Sensitivity,specificity,coincidence rate and ? value of HGIN diagnosis by pCLE were 95.65%(95%CI,76.03%-99.77%),98.25%(95%CI,89.37%-99.91%),97.50%and 0.94.2.Molecular imaging of gastric cancer by CLE It was confirmed that there was a low HER2 expression level in MGC-803 and a high HER2 expression level in NCI-N87 by western blot.AF-anti-HER2 as a molecular probe could bind to HER2 over expression cell easily and specifically.The proper experimental condition to observe the fluorescence was that the tumor-bearing mouse reveived 4?g AF-anti-HER2 through caudal vein injection and was fed away from light for 24 hours.There were eight NCI-N87 tumor-bearing mice with AF-anti-HER2 having 3+ or 4+ fluorescence grading,one NCI-N87 tumor-bearing mice with AF-anti-HER2 hasing 2+ fluorescence grading,and four NCI-N87 tumor-bearing mice with AF-IgG having 2+ fluorescence grading or lower grading,which was coincident with the result of immunohistochemistry.There were three MGC-803 tumor-bearing mice with AF-anti-HER2 having 4+ fluorescence grading,four MGC-803 tumor-bearing mice with AF-anti-HER2 having 2+fluorescence grading or lower grading,four MGC-803 tumor-bearing mice with AF-IgG having 2+ fluorescence grading or lower grading,which was coincident with the result of immunohistochemistry.Besides,there was one MGC-803 tumor-bearing mouse withAF-anti-HER2 hasing low fluorescence grading but a high HER2 expression level in immunohistochemistry.Conclusions:Diagnosis of AG,GIM,LGIN and HGIN can be made by pCLE morphological imaging,so is the GIM subtype differentiation.Using AF-anti-HER2 as a molecular probe,pCLE can achieve specific molecular images to show HER2 expression in xenograft properly.Significance:The study shows that Diagnosis of AG,GIM,LGIN,HGIN and GIM subtype differentiation can be made by pCLE morphological imaging,which supports the clinical application of pCLE.Physicians can get a targeted biopsy accoding to the pCLE diagnosis,which can improve detection rate and reduce misdiagnosis rate.HER2 molecular imaging of gastric cancer by pCLE was studied in gastric cell lines and tumor-bearing mice for the first time.The result shows that pCLE will play an important role in gastric cancer molecular diagnosis,prediction and monitor targeted therapeutic effect.