Women were not eligible to participate if they

Women were not eligible to participate if they normally had a previous or concurrent malignancy including hematologic malignancies, were receiving active chemotherapy, or had chemotherapy or steroid-based therapy in the past 6 months. Women undergoing immunosuppressive treatments or women with an autoimmune disorder were also excluded. A positive breast cancer diagnosis was defined as women having invasive ductal or lobular carcinoma and ductal carcinoma in situ as verified by pathologic evaluation. Women diagnosed with either fibroadenoma, fibrocystic changes (including sclerosingadenosis, and benign papilloma), atypical ductal hyperplasia, atypical lobular hyperplasia and lobular carcinoma in situ were included in the control (healthy) group. As stated, to determine the clinical status of the women, a biopsy confirmation was required.

The control group had negative biopsy samples and was not dependent upon imaging interpretations. In situations where there was a difference between the results of the needle biopsy and surgery, the pathologic findings at surgery overruled the needle biopsy results. Verification of a definitive breast cancer diagnosis was dependent on both imaging and pathology concordance. Patients with biopsy samples with no pathological report, or with no final diagnosis, were excluded from the study. A total of 546 samples were obtained from five centers worldwide (Israel, Italy, and the USA) (Table 1). All blood samples were collected with local Institutional Review Board (IRB) approval after each participant signed an informed consent.

The trial registration ID is NCT00331942. Table 1 Study population information��number of samples collected at each site according to final diagnosis as verified by biopsy of the lesion. Patients were considered cases with either invasive cancer or DCIS and ADH, ALH, LCIS, and other lesions were … Plasma was collected from whole blood using heparin tubes (cat. No. 455084, Greiner Bio-One, Frickenhausen, Germany) centrifuged at 3,000 �� g for 10 min at room temperature (RT), and aliquots were stored frozen at ?80oC until ELISA analysis. At the MD Anderson Cancer Center only, plasma was centrifuged at 1,300 RPM for 30 min at 4oC and aliquots were stored frozen at ?80oC until ELISA analysis. Data forms were completed by each site to obtain clinical information and final pathological diagnosis.

Antigen selection for AAb assay Antigens were chosen from the current literature according to their known involvement in the humoral response against breast cancer (Table S1 in Supplementary Data on-line��Details of antigens used in this study). An initial set of 15 different antigens, all showing the ability to elicit antibody production in breast cancer patients (and some, to a GSK-3 smaller extent, in healthy populations as well) were chosen for initial testing (Table 2). All proteins and peptides were purchased from different suppliers (Table S1 in Supplementary Data on-line).

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