The BC-APPS1 study
The BC-APPS1 was a single-arm, single-centre phase II study registered under the name ‘a pilot prevention study of the effects of the anti-progestin ulipristal acetate (UA) on surrogate markers of breast cancer risk’ (EudraCT registration number: 2015-001587-19; registration date: 15 July 2015; Greater Manchester-South, Research Ethics Committee number 15/NW/0478). Eligible women were premenopausal, 25–45 years of age with regular menses and a residual lifetime breast cancer risk of at least 17% (≥1:6) assessed by the Tyrer–Cuzick risk estimation programme (v7.02; https://ems-trials.org/riskevaluator/). All women were recruited from the Family History Risk and Prevention Clinic at the Nightingale Centre, Wythenshawe Hospital, Manchester, UK. Complete eligibility criteria are provided in the protocol (Supplementary Appendix 1). Following informed consent and screening, participants underwent blood testing to confirm serum progesterone levels consistent with the luteal phase of the menstrual cycle (15 nmol l−1 or more; Abbott Architect Immunoassay) and then underwent a contrast-enhanced MRI scan of both breasts (Philips Achieva 1.5 T MRI). A baseline VAB was then performed by a consultant radiologist under ultrasound guidance to identify areas of fibroglandular breast tissue. On the onset of menstruation, participants commenced 5 mg oral tablets of commercially available UA, taken once daily for a duration of 12 weeks. During the final week, blood was drawn for progesterone levels, MRI of both breasts was repeated and VAB of the contralateral breast was performed. For each VAB, 10 cores were taken with a 10-G biopsy needle (cores were divided and fixed in formalin for paraffin embedding, snap frozen for RNA extraction and placed in tissue culture medium for subsequent digestion to single-cell suspensions). The primary end point was the change in epithelial cell proliferation measured by the percentage of Ki67 staining before and after treatment. Secondary end points were (1) percentage of luminal, basal and mixed colonies by morphological analysis of the adherent feeder layer assay; (2) percentage of luminal progenitor cells (EPCAM+CD49f+) by flow cytometry analysis; (3) tissue stiffness assessed as the reduced indentation modulus by atomic force microscopy; (4) mean tissue section percentage fibrillar collagen assessed by PSR staining and polarized light microscopy; (5) background parenchymal enhancement assessed by MRI; (6) the side effect profile of UA assessed by CTCAE (v4.03); and (7) the relative change in Ki67 with UA treatment between those with and without known mutation in BRCA1/2 genes. Exploratory end points are included in the attached protocol and the methods described below. All participants had blood drawn for complete blood count, renal function and liver function tests at baseline and 3 months. Treatment with UA was suspended by the EMA/MHRA in February 2018 due to concerns of liver damage by UA. On reopening of the study after the suspension was lifted, the protocol was amended to include measurement of liver function tests every 4 weeks during treatment and a final check 4 weeks after the end of treatment. Toxicity assessment was undertaken every 4 weeks using CTCAE (v4.03).
Other human breast tissue procurement
Normal breast tissue samples were collected from women at moderate or high risk undergoing surgical risk-reducing mastectomy at the Manchester University NHS Foundation Trust through the Manchester Cancer Research Centre Biobank. Fully informed consent from all patients was obtained in accordance with local National Research Ethics Service Guidelines, and the collection of demographic and clinical data was granted under the MCRC Biobank Research Tissue Bank Ethics (NHS NW Research Ethics Committee 18/NW/0092).
Frozen primary human female breast tissue was additionally sourced from the Breast Cancer Now Tissue Bank (REC 15/EE/0192), with all procedures conducted in compliance with applicable ethical regulations.
Breast tissue processing
Normal breast tissue obtained via VAB (mean tissue weight of 1.22 g; 95% CI 1.06–1.37) was manually minced with a scalpel into small fragments (approximately 2 mm3 pieces) and incubated in dissociation medium: phenol red-free DMEM/F12 with HEPES (Gibco) supplemented with 25% BSA Fraction V solution (Gibco), 1 mg ml−1 collagenase/hyaluronidase (Stem Cell Technologies) and 5 μg ml−1 insulin (Sigma). After overnight digestion at 37 °C with shaking at 100 rpm, the dissociated breast cell suspension was centrifuged at 450g for 5 min at 4 °C. The fat layer was discarded and the epithelial pellet was resuspended in DMEM/F12 medium and centrifuged again. This wash step was repeated until the supernatant became clear. Then, 1 ml of pre-warmed 0.05% trypsin-EDTA was added to the enriched epithelial pellet, pipetting it up and down gently with a P1000 pipette for 2–3 min. Next, 10 ml of cold Hank’s balanced salt solution (HBSS; Gibco) supplemented with 10 mM HEPES (Gibco) and 2% FBS (Gibco) was added and the cells were centrifuged at 450g for 5 min at 4 °C. After removing the supernatant, 1 ml of pre-warmed 5 mg ml−1 dispase (StemCell Technologies) was added to the sample and pipetted for 1 min to further dissociate cell clumps. Cells were resuspended in HBSS–HEPES–FBS solution, centrifuged and supernatant was discarded. The HBSS–HEPES–FBS solution was then added and cells were sieved using 100-μm and 40-μm filters to yield a single-cell suspension. Cells were counted using a Fuchs Rosenthal haemocytometer (mean cell yield of 1.39 million; 95% CI 0.99–1.79) and plated for experiments. Remaining cells were frozen using Bambanker freezing media (Lymphotec Inc.) until further analysis (for example, flow cytometry and scRNA-seq).
Normal breast tissue obtained via risk-reducing mastectomy was cut into 2–3 mm3 pieces and digested overnight at 37 °C with collagenase IA (C2674, Sigma) and hyaluronidase (H3506, Sigma), both to a final concentration of 1 mg ml−1, in phenol red-free DMEM/F-12 medium (Gibco). Following enzyme digestion, the breast tissue was washed three times with medium by centrifuging at 400g for 10 min and discarding the supernatant. The pellet was resuspended in medium and left to sediment three times for 25 min at 4 °C on a flat surface. The collected breast organoids or microstructures from each tissue preparation were frozen in Bambanker freezing medium until experimental use.
Measurement of progesterone (P4) levels
Serum progesterone levels were determined at baseline and after 3 months of anti-progestin treatment. Serum progesterone concentrations were measured by an NHS-accredited laboratory using the Abbott Architect immunoassay (Abbott Laboratories).
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