Two millilitres of ethylene diamine (EDTA) blood samples were collected from 358 breast cancer patients and 363 healthy females from September 2016 to March 2018. All cancer cases were staged according to the revised TNM classification criteria36 by a pathologist. All healthy females were recruited from patients without immune disorders, chronic diseases, and family history of cancer and negative mammography examination results. The mammogram results of all participants were graded by a radiologist. EDTA blood samples were provided by the National Cancer Institute, Thailand and King Chulalongkorn Memorial Hospital. All samples were centrifuged for 15 min at 3000 rpm to separate the WBC layer (buffy coat). Then, 100 µl of the WBC layer was transferred to a 1.5-ml Eppendorf tube. All subjects participating in blood collection were given a self-administered questionnaire to collect their medical history, which was carefully recorded. All samples were obtained under a protocol approved by the Ethics Committee, Faculty of Medicine, Chulalongkorn University, Thailand (approval number: IRB 034/59), and National Cancer Institute, Thailand (approval number: 157_2016RC_OUT487). Thai Clinical Trials Registry number is TCTR20180804002. The collection of blood samples from all participants was performed based on the WHO guidelines. This study was conducted in accordance with the Declaration of Helsinki. Supplementary data 2 provides the STROBE checklist. Signed informed consent was obtained.

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