This study aimed to determine whether three pre-analytical variables - fixation duration, cold ischemia time, and fixative preparation (4% neutral buffered versus 4% non-buffered formaldehyde) - impact the preservation and evaluation of tissue biomarkers in invasive breast cancer.<h4>Methods</h4>We conducted an exploratory, proof-of-concept, experimental study using fresh mastectomy tissue from a 34-year-old patient with invasive ductal carcinoma (pT4, hormone receptor-positive, HER2-negative, Ki67 = 40%) who had not received neoadjuvant chemotherapy. Here, NR4A1 is linked to invasive breast carcinoma.