ESCC serum samples were obtained from newly diagnosed ESCC patients who underwent surgery between December 2014 and August 2017 in Fujian Cancer Hospital & Fujian Medical University Cancer Hospital. All enrolled patients underwent face-to-face interviews by trained interviewers using a standardized questionnaire. We recruited subjects who underwent surgery at Fujian Cancer Hospital & Fujian Medical University Cancer Hospital. Pathological results were available for all patients with ESCC, and the results were confirmed by pathologists in this hospital. We excluded patients with other cancers in this study. All patients did not receive chemotherapy or radiotherapy before they underwent surgery. Tumor stages were identified according to the American Joint Committee on Cancer tumor node metastasis (TNM) staging criteria. In preliminary screening, the exosomal circRNA was extracted from ESCC patients with lymph node metastasis (n=4), sex- and age-matched (±5 years) ESCC patients without lymph node metastasis (n=4) and normal control individuals (n=4). In this study, the expression profiles of exosomal circRNA were analyzed using microarray analysis and compared in terms of three groups (A group: ESCC with lymph node metastasis vs ESCC without lymph node metastasis; B group: ESCC with lymph node metastasis vs normal control individuals; C group: ESCC with lymph node metastasis vs postoperative ESCC with lymph node metastasis).

Samples were categorized into the ESCC with lymph node metastasis group (LNM group) if they came from patients who had lymph node dissection ≥ 10 and lymph node metastasis ≥ 1. Samples were categorized into the ESCC without lymph node metastasis group (non-LNM group) if they came from patients who had lymph node dissection ≥ 10 and lymph node metastasis =0. Blood sampling was performed before surgery. In addition, postoperative blood samples were also collected from patients with ESCC. Samples from normal control individuals were retrieved from four physical examinations, which excluded cancer and inflammation, in the same hospital. A total of 69 serum samples from patients with ESCC were collected. The clinical characteristics of the ESCC patients are summarized in Supplement Table 1. Among the patients, there were 35 patients with lymph node metastasis and 34 patients without lymph node metastasis. This study was approved by the Ethical Committee of Fujian Medical University (number: 201495), and the subjects provided informed consent.

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