68-5

50/120

500discovery of esophageal carcinomas in its early stage remains difficult. Clinical biomarkers and tests for early detection of esophageal carcinomas have not yet been established. Developments of non-invasive method for early diagnosis with better sensitivity and specificity are expected.In recent decades, various scent detection studies have been performed with animals and electronic devices5). The presence of some infections and malignant tumors can be identified based on changes in patients’ metabolites. Devices for detecting vola-tile organic compounds (VOCs) that are odorous elements of cancers are also being developed6, 7). McColloch et al. reported high accuracy rates using the exhaled breath of dogs with lung and breast cancers8). Breath analysis using gas chromatog-raphy has also identified several VOCs specific to lung and breast cancers9-11). The identification of cancers by exhaled breath analysis is expected to develop as non-invasive methods for detection of early-stage cancer. Recent studies demonstrated the clinical potential of VOCs profiling to identify esophageal or gastric adenocarcinoma12, 13). To date, there have been no reports of VOCs specific to ESCC, and the present pilot study aimed to identify just such VOCs as clinical biomarkers.Materials and MethodsSubjectsExhaled breath was collected from 17 patients who had been diagnosed with ESCC and 9 healthy volunteers in Juntendo University Hospital between July 2012 and November 2013. Healthy volunteers were selected from staffs at our facility who had been approved to participate in the study and who had no history of cancer or other medical condi-tions. No upper gastrointestinal endoscopy was required. The exhaled breath was analyzed for its VOC contents. All subjects were interviewed to confirm the disease for which they were being treated, whether they were taking any medications or smoked, and their family histories. Patients who were being treated with either radiotherapy or chemotherapy were excluded from the study, since such treatments might alter their metabolism. All protocols were approved by the ethical committee of the Juntendo University Hospital (No.16-062), and all participants provided written informed consent before their participation in this study according to the guidelines established in the Declaration of Helsinki.Exhaled breath collectionAll participants were fasted for 6-8 hours prior to their breath sample collection. For each subject, 1L of exhaled breath was collected in a Supel™ Inert Gas Sampling Bag (Sigma-Aldrich Co. LLC. MO, USA) in a clean-air environment before any treatment. Prior to the collection, each sampling bag was washed with nitrogen to reduce other influences. All collected samples were stored and processed in a cold room at 4℃.Extraction and analysis of volatile organic compoundsSamples were analyzed by gas chromatography- mass spectrometry (GC-MS) combined with solid phase micro extraction (SPME) (Sigma-Aldrich Co. LLC. MO, USA). The SPME was used for pre- concentration of VOCs in the breath samples. A manual SPME holder with 85μm Carboxen®/Polydimethylsiloxane (CAR/PDMS) fiber (Sigma- Aldrich Co. LLC. MO, USA) was inserted into the Supel™ Inert Gas Sampling bags for 16 hours at room temperature. The VOCs extracted in SPME fiber were desorbed thermally in the heated GC injector at 250°C (splitless mode) and analyzed by GC-MS. The GC-MS analysis was performed with a TRACE GC connected to TSQ QUANTUM GC (Thermo Fisher Scientific Inc., MA, USA). A CP Pora PLOT Q capillary column (0.25mm i.d x 25m x 8μm firm thickness, Agilent Technologies Inc, CA, USA) was used for separation of VOCs. The column temperature started at 40°C for 3 minutes, then raised at 10°C per minute to 250°C and held for 5 minutes. Helium was used as carrier gas at 1ml/min. MS transfer line was set at 250°C. The MS analysis was carried out Electron impact ioniza-tion (EI) mode with 70eV of electron energy at scan range m/z:10-400. Ion source temperature was set at 250°C. The detected VOCs were identi-fied by NIST MS Search 2.0 (Thermo Fisher Scien-tific Inc., MA, USA).Statistical analysisAfter alignment the comprehensive peaks in GC-MS chromatograms of every sample using Thermo Scientific SIEVE Software (Thermo