Definition of carcinoma at the EGJ
We defined cancer at the EGJ according to the Japanese classification [20]. In this classification, the area extending 2 cm above to 2 cm below the EGJ is designated as the EGJ area. The location of an EGJ carcinoma is described using the symbols E (proximal 2 cm segment) and G (distal 2 cm segment), with the dominant area of invasion described first; i.e., E, EG, E = G (both areas equally involved), GE, or G.
Enzyme-activatable fluorescent targeting agent
The EP-HMRG probe was purchased from Goryo Chemical (Sapporo, Japan), resuspended in 10 mM dimethyl sulfoxide (Sigma-Aldrich, St. Louis, MO, USA) and then stored at -80°C. Before use, the EP-HMRG suspension was thawed to room temperature and diluted to 100 μM with phosphate-buffered saline (PBS, Life Technologies, Carlsbad, CA, USA).
Patients
This study prospectively reviewed early EGJ adenocarcinoma resected by ESD in 21 patients at five hospitals between May 2016 and June 2018. All ESD procedures were performed by experienced endoscopists.
The resected specimen was immediately extended on a black rubber mat and fixed with pins, and then 100 μM EP-HMRG was sprayed onto the specimen. Fluorescence imaging was performed using a handheld fluorescence imaging system (Discovery; INDEC Medical Systems, Santa Clara, CA, USA) that captured white-light images and fluorescence images with 450–490 nm blue excitation light. The fluorescence images were recorded every minute for 10 minutes after the EP-HMRG administration. Subsequently, the specimens were washed with PBS and observed using an endoscope (H290Z, Olympus, Tokyo, Japan) under white light.
The fluorescence intensities were measured with ImageJ software (National Institutes of Health, Rockville, MD, USA). We set regions of interest (ROIs) at the area with the most fluorescence signal in the tumor lesion and in the non-tumor region adjacent to the tumor lesion. The mean fluorescence intensity of each ROI was measured as pixel intensity values ranging from 0 to 255, and the contrast-to-background ratio (CBR) was also measured.
Ethics statement
The Ethical Review Committee of each hospital approved this ex vivo clinical study protocol. All patients provided informed consent to participate in this study.
Pathological examination
Specimens were fixed in 40 g/L formaldehyde saline, embedded in paraffin, and cut into 5-μm sections. Tissue sections were stained with hematoxylin and eosin and then microscopically examined for the histological type, tumor size, depth of invasion, lymphovascular invasion, and resected margin by an experienced pathologist (KCH), according to the World Health Organization classification. Immunohistochemical analysis of DPP-IV expression was performed using an anti-DPP-IV antibody (Novus Biologicals, Littleton, CO, USA). The subtype of intestinal metaplasia in the non-tumor region was determined using the MUC5AC (Agilent, Santa Clara, CA, USA), MUC6 (Abcam, Cambridge, UK), MUC2 (Spring Bioscience, Pleasanton, CA, USA), and CD10 (Agilent) expression patterns. MUC5AC and MUC6 are markers of the gastric phenotype, whereas MUC2 and CD10 are markers of the intestinal phenotype. We defined the complete type as decreased expression of gastric mucin (MUC5AC or MUC6) and co-expression of MUC2 and CD10. The incomplete type was defined as the expression of MUC5AC or MUC6 and MUC2 [21].
Statistical analysis
Receiver operating characteristic (ROC) curves were used to determine the sensitivity, specificity, and accuracy. All analyses were performed using GraphPad Prism version 6 (GraphPad Software, San Diego, CA, USA).