The data collection process of the cadavers included patients' profile, gland characterization, lesion size, histological type, and molecular surrogates. Cadavers profile included age, ethnicity, comorbidities, medications, cause of death, breast screening adhesion, and breast cancer risk factors. Gland's characteristics included dimensions, weight, and size.
The quantitative statistical method of analysis was based on the overall multi-dimension constructs measurements for every factor, descriptive statistics, regression, and parametric as well as non-parametric tests. The regression statistics was used to determine the correlation between the multi-dimension construct assessment and each factor.
The study group consisted of a series of consecutive medico-legal autopsies on fresh Portuguese cadaver performed from July 2016 to December 2019 at the National Institute of Legal Medicine and Forensic Science, Lisbon, Portugal.
The criteria for exclusion were age younger than 40 years, the autopsy performed in less than 48 hours after death, extensive injury to one or both breasts, and known or clinically evident breast cancer. Once the eligibility criteria were met, and the sample collection authorization was obtained, a bilateral subcutaneous modified radical mastectomy (bsMRM) was performed through a Douformentel incision (allowing the subsequent reconstruction, previous to corpses release) in each fresh cadaver at the National Institute of Legal Medicine and Forensic Science.
General information, such as age, height, weight, and body mass index (B.M.I.), was obtained from the cadaver's referring file when available, while past medical history data was not included due to inadequate collection.
Each specimen was properly identified in means of spatial orientation and, after conditioning in sealed bags, was transported within an appropriate container to the Hospital São Francisco Xavier (Lisbon, Portugal), and submitted to measuring (three- dimensions), waiting, inspection, palpation, ultrasound, and mammography by breast radiologists and breast surgeon.
The collected tissues were imaged using the G.E. Healthcare digital mammography system, Senographe EssentialTM (G.E. Healthcare Bio Sciences, Pittsburgh, PA, U.S.A.), with an X-ray beam of 27 kV (range, 60 70 mA) and 10 15 decanewtons (daN) compression, depending on tissue density and size. The visualization screen had a resolution of five megapixels (G.E. Healthcare LOGIQTM S7 Expert ultrasound system, with a medium frequency of 9 15 MHz; G.E. Healthcare Bio Sciences).
Breast tissue, classified as Breast Imaging Reporting and Data System (BI-RADS) category three or higher, was submitted to wire-guided or direct excisional surgical biopsy by the author. According to the 5th edition of the ACR BI-RADS Atlas, ACR BI-RADS (ACR, s.d.) system has been used.
In the pre-analytical phase, breast biopsies were fixed in 10% buffered formalin (JTBaker) for 24 hours, and lumpectomy specimens were fixed for 48 to 72 hours at room temperature (20°C). Formalin-fixed, paraffin (VWR International, EUA) embedded tissues were processed in Sakura's "Tissue-Tek VIP" and cut into 3 μm sections, one cut per adhesive slide (Superfrost Plus Gold - Thermo Scientific, EUA), with respective positive control. Tissue section adhesion time and temperature were held constant for 1 hour at 70oC.
Following these procedures, the slide was subjected to labeling by the immunocytochemistry (ICC) method.
The ICC panel of primary antibodies used against Ki67 (clone 30-9, Cat. 790-4286), ER (clone SP1, Cat. 790-4324), and PR (clone 1E2, Cat.790-2223) were performed in the BenchMark ULTRA using Optiview DAB IHC Detection Kit (Cat. 760-700), for Ki67 and Ultraview Universal DAB Detection Kit (Cat. 760-500), for ER and PR, all from Ventana Medical Systems, Tucson, USA.
The slides were observed by a surgical pathologist under an optical microscope.