| Backgrounds and aims:Helicobacter pylori infection occurs in approximately half of the world population, especially in developing countries. On the basis of epidemiological and experimental observations, H. pylori infection almost invariably causes chronic inflammation of the gastric mucosa, and this infection is a crucial factor in the multistep carcinogenic process of gastric cancer. H. pylori has been cited as a class I gastric carcinogen in 1994.H. pylori infection can be diagnosed by noninvasive and invasive methods. It is important to note that the selection of the appropriate test depends on the clinical setting. Histological analysis of biopsy specimens provides good sensitivity and specificity, but the special stains add much time and expense to the diagnostic evaluation. Therefore, diagnosis of H. pylori infection during ongoing endoscopy would be very helpful to obtain an immediate diagnosis. Recently, confocal laser endomicroscopy (CLE) has been developed to realize in vivo histology. CLE combines standard videoendoscopy with confocal microscopy imaging of gastrointestinal mucosa during endoscopy. Acriflavine guided endomicroscopy was used for the first time to detect H. pylori in a patient in 2005. However, the diagnostic efficacy of CLE for H. pylori infection lacks detailed data. Taking into account that H. pylori is an important causative factor in gastric carcinogenesis, it is logical to assume that its eradication may have an important role to play in the prevention of gastric cancer. However, although H. pylori eradication causes regression of inflammatory changes in the gastric mucosa, it remains unclear whether this prevents gastric cancer. In particular, the effect of H.pylori eradication in patients with intestinal metaplasia (IM) is highly uncertain, which hinders clinical decisions making in practice. Eradication therapy is not beneficial if it is given to individuals beyond the "point of no return". The discrepancies are partly due to the patchy distribution of IM, IM is difficult to assess using limited random biopsy specimens, leading to imprecise results as a consequence of sampling errors. Endomicroscopy can identify gastric IM with high accuracy, fluorescein sodium is a most widely used contrast agent during endomicroscopy. Furthermore, it is also a small moleculethat is widely used as hydrophilic marker for paracellular permeability studies. An impaired mucosal barrier integrity may be involved in the carcinogenic processes. CLE can provide histological and functional imaging in different gastric mucosa.The association between Helicobacter pylori infection and development of gastric cancer is well established. In Correa’s model of carcinogenesis of intestinal type gastric carcinomas, chronic gastritis slowly progresses through the premalignant stages of atrophic gastritis, intestinal metaplasia and dysplasia to gastric adenocarcinoma. Endoscopic mucosal resection (EMR) has been widely accepted as a treatment modality for early malignant lesions. Although the therapeutic efficacy of EMR has been validated, standard EMR techniques are associated with risks of recurrence, especially when resections are not performed en bloc, or when the residual neoplastic tissue is present on the resection margins. The success of EMR implementation not only depends on the depth of invasion but also the lateral spread of the lesion. If the adequacy of resection could not be determined, the completeness of resection margin had to be assessed by intensive follow-up endoscopy with multiple biopsies. The value of CLE in predicting completeness of EMR has not been explored.Therefore, the aims of this study were as follows:1. To compare CLE features of H. pylori infection with histology findings and evaluated the diagnostic efficacy of CLE for in vivo diagnosis of H. pylori infection.2. To evaluate the gastric paracellular barrier changes of non-metaplastic mucosa and metaplastic mucosa before and after eradication of H. pylori.3. To prospectively assess the feasibility and efficacy of CLE in predicting complete resection after EMR.MethodsPart I:Confocal endomicroscopy for diagnosis of H. pylori infectionAll procedures involved use of a acriflavine guided confocal laser endomicroscope (Pentax EC-3870K). A total of 103 consecutive patients scheduled to undergo endoscopy were enrolled from August 2008 to March 2009. The first 20 patients were recruited for a pilot study. The CLE recording images and corresponding histopathology images were openly evaluated by 3 senior endoscopists and 1 pathologist, and CLE image criteria for H. pylori infection were established. Then 83 consecutive patients were prospectively evaluated, and data were correlated with final diagnosis of H. pylori infection in a blinded manner.15ml acriflavine hydrochloride (0.05%) was applied topically by use of a spray catheter. The greater and lesser curvature of the antrum and corpus were carefully observed separately on CLE. We defined a case as H. pylori infection with positive rapid urease test and Giemsa staining results.PartⅡ:Effects of H. pylori infection and its eradication on epithelial barrier in patients with gastric intestinal metaplasiaConsecutive outpatients with previous histologically confirmed IM of the gastric mucosa were recruited for endoscopic surveillance, from January 2009 through November 2009. After being informed about the purpose, those who were willing to choose CLE instead of conventional endoscopy were included in the study. Endomicroscopy was performed as described previously using intravenous fluorescein as a contrast agent. Specific metaplastic mucosa and non-metaplastic mucosa were identified in vivo, and 2 targeted biopsy samples each were taken from the metaplastic areas and non-metaplastic areas (one for histological analysis and the other for electron microscopy). For electron microscopy, at least 50 junctions were examined. Results are expressed as percentage of "leaky" junctions with paracellular invasion of lanthanum nitrate. Post-CLE assessment of paracellular permeability was devised, The proportion of these "leaky" cells among surface epitheliums was graded semiquantitatively on a 0 to 4 scale and this CLE score system was validated by electron microscopy. H. pylori infected patients received a 2-week eradication treatment, CLE with target biopsies were repeated after 6 months.Part III:Confocal endomicroscopy for in vivo prediction of completeness after endoscopic mucosal resectionConsecutive patients with gastric neoplastic lesions who underwent EMR at our endoscopy unit were included between January 2009 and April 2010. Before EMR, Endoscopic ultrasound (EUS) was obtained for all patients to assess the depth of invasion and lymphadenopathy. EMR was performed by using cap-assisted or "inject and cut" resection techniques by standard video endoscope. Complete remission was defined as:Clear margin specimens and one negative follow-up endoscopy, or for the patients with piecemeal resection or indefinite marginal status, two negative follow-up endoscopies with multiple biopsies were required. Patients with remnant tissue in vertical margin were defined as having treatment failure, and operable patients were scheduled for gastrectomy. Two weeks after EMR, the circumferential margins of the defect were inspected by using CLE, and completeness of excision was predicted from the CLE image. Additional EMR were performed if necessary. The in vivo CLE diagnosis was validated against the final histopathology. ResultsPartⅠ:We found good association between histopathology and CLE findings. H. pylori infection was identified by CLE with any of the following 3 features:white spots, neutrophils and microabscesses. A total of 83 patients were enrolled in this phase.37 patients (44.6%) were positive for H. pylori infection. In total,6823 CLE images were acquired (mean 82.2 images per patient). The accuracy, sensitivity and specificity of CLE diagnosis of H. pylori infection were 92.8%,89.2% and 95.7%, respectively. The meanκvalue for interobserver agreement in the prediction of H. pylori infection was 0.78. Neutrophils was the best diagnostic feature and had good sensitivity (83.8%) and specificity (97.8%). H. pylori-associated changes were more common in the antrum than in the corpus among infected patients (P<0.001).PartⅡ:Of the 42 patients enrolled in the study, all the metaplastic sites sampled for electron microscopy was diagnosed as IM by histopathology. H. pylori infection was present in 20 (47.6%) subjects. The percentage of impaired paracellular junctions was significantly increased in H. pylori-positive samples (54±31%,72±28%) and H. pylori-negative IM samples (67±34%) compared with H. pylori-negative control samples (3±6%, all p<0.05). In the post-CLE assessment, the mean confocal scores of H. pylori-negative gastric epithelium were 0.21±0.24. This score was significantly increased in H. pylori-negative IM epithelium (2.82±1.00, p<0.05) and H. pylori-positive epithelium (2.60±1.03,2.83±0.81, both p<0.05) compared with H. pylori-negative epithelium. Post-CLE assessment correlated well and linearly with the electron microscopy findings (R-squared= 0.834; p<0.0001). After 6 months, H. pylori infection was successfully eradicated in 14 patients. Of these patients, paracellular barrier dysfunction of non-metaplastic mucosa was significantly improved (both p<0.001). However, there was no significant changes in IM mucosa despite the treatment.PartⅢ:Twenty-seven lesions were removed by EMR in 27 patients. After excluding 3 patients for gastrectomy, a total of 24 patients underwent CLE assessment, of which 9 patients with indefinite lateral margins underwent at least two consecutive CLE follow-up. A total of 19 lesions were regarded as complete remission, and 5 lesions (two high-grade intraepithelial neoplasia and three low-grade intraepithelial neoplasia) were incompletely excised according to the final pathologic diagnosis. After CLE evaluation, seven of 24 patients (29.2%) were suspected to have neoplasias at the ulcer margin during CLE according to the gland polarity and epithelial thickness. The accuracy of CLE in predicting incomplete resection for original lesions was 91.7%, the sensitivity and specificity were 100.0% and 89.5%, respectively. Six patients with residual lesions were treated by additional EMR guided by CLE. There was no recurrence on endoscopic biopsies at a mean follow-up of 8.3 (range 4-15) months.Conclusions1. H. pylori infection can be identified by specific cellular and subcellular changes of the surface gastric mucosa with CLE.CLE is a simple, rapid, and useful method for predicting H. pylori infection in vivo during endoscopy.2. H. pylori infection and intestinal metaplasia is associated with an impaired paracellular barrier integrity, and this disruption may be unreversible after H. pylori eradication. Fluorescein leakage can be used as a molecule marker for paracellular permeability studies in vivo.3. Confocal laser endomicroscopy has a high accuracy for prediction of remnant tissue after EMR, the target therapy can potential improve the cure rates of endoscopic therapies.SignificanceThe practical CLE criterias of H. pylori infection and gastric metaplasia were established and validated by prospective studies. The CLE procedure can provide a "smart" biopsy rather than random biopsies. We demonstrated that disruption of gastric paracellular barrier were not only observed within H. pylori infected mucosa but also were present in IM mucosa for the first time, and the dysfunction of IM seems difficult to recover. These findings were helpful to perform targeting H. pylori eradication therapy in patients with different mucosal changes, and suggest that disruption of the paracellular barrier may provide potentially important insights into early events in gastric carcinogenesis. Fluorescein leakage is hopeful to apply in the assessment of gastric permeability. EMR in combination with post-assessment by CLE is an efficacious strategy for the treatment of gastric neoplastic lesions. CLE may lead to significant improvements in the clinical surveillance after endoscopic resection. |
PDF Full Text Request