Clone sp1

Formalin-fixed paraffin-embedded tissue was collected from the archives of the pathology division of our hospital. Hematoxylin–eosin archival slides were revised by an expert pathologist and the diagnosis was confirmed in all cases. The most representative slide of each sample was selected for IHC in cases with available material. Specifically, three-micron-thick serial paraffin sections of each case were processed by IHC using an automated immunostainer (Ventana BenchMark Ultra AutoStainer, Ventana Medical Systems, Tucson, AZ, USA) with antibodies against p53 (clone DO-7, catalogue number 7800-2912,Ventana Medical Systems, Tucson, AZ, USA), ER (clone SP1, catalogue number 790-4324, Ventana Medical Systems, Tucson, AZ, USA), PgR (clone 1E2, catalogue number 790-2223, Ventana Medical Systems, Tucson, AZ, USA), and the MMR status, evaluating the protein expression of MLH1 (clone M1, catalogue number 760-5091, Ventana Medical Systems, Tucson, AZ, USA), PMS2 (clone EPR3947, catalogue number 760-5094, Ventana Medical Systems, Tucson, AZ, USA), MSH2 (clone G219-1129, catalogue number 760-5093, Ventana Medical Systems, Tucson, AZ, USA), MSH6 (clone 44, catalogue number 760-5092, Ventana Medical Systems, Tucson, AZ, USA). Appropriate positive controls were included for each IHC run.

Firstly, the fresh tissues were fixed in 10% buffered formalin (PH 7.0) and embedded in paraffin. The paraffin-embedded tumor samples were then sectioned continuously into 4-μm-thick sections. Then the sections were dewaxed in xylene and rehydrated in graded alcohols. A normal rabbit IgG antibody was used as a negative control. Following antigen retrieval by microwave heating (95°C for 20 min), sections were then incubated with ESR1 (Clone SP1, Ventana Medical Systems. Inc.) at 4°C overnight. After washing, a horseradish peroxidase-labeled goat antibody against a mouse/rabbit secondary antibody (Envision; Dako, Glostrup, Denmark) was added, followed by incubation at room temperature for 30 min. The signal was then detected with 3,3′-diaminobenzidine tetrahydrochloride (DAB). Two independent pathologists blinded to the clinical information scored the ESR1 expression levels in tumor cells by assessing (a) the proportion of positively stained cells (0, < 5%; 1, 6–25%; 2, 26–50%; 3, 51–75%; 4, >75%) and (b) the signal intensity (0, no signal; 1, weak; 2, moderate; 3, strong). The score was the product of a × b. Considering that the number of patients with score > 0 in ESR1 was very low, we used dichotomic classification (positive/negative). Therefore, a positive group (a × b>0) and a negative group (a × b = 0) were used (Figure S1).

Immunohistochemical analysis of the tumors treated with trastuzumab (n = 2) or vehicle (n = 3) was performed to confirm the changes in ER expression observed by [¹⁸F]FES -PET. The immunohistochemical staining of the formalin-fixed paraffin-embedded SKOV3 tumors was performed according to the routine protocol for clinical samples. ER-α rabbit monoclonal primary antibody (Clone SP-1, Ventana) and a c–erbB-2/HER-2/neu rabbit monoclonal antibody (Clone SP-3, Thermo Fisher scientific) were used for immunohistochemical staining of ER-α and HER2, respectively. HER2 and ER immunohistochemistry results were analyzed by an expert histopathologist according to a 4-level scoring system: 0, 1, 2, and 3+ for no, weak, moderate, and high-intensity staining, respectively.

Immunohistochemical analysis on BC specimens were performed at our Pathology Department and tumors were defined as TNBC if ER (Ventana, Clone SP1, pre-diluted) and PgR (clone 30-9; Ventana Medical Systems Inc, pre-diluted) IHC staining was ≤ 1%, together with 0/1 + score HER2 on IHC (clone 4B5; Ventana Medical Systems Inc, pre-diluted) and/or non-amplified on fluorescent in situ hybridization (FISH) for 2 + score HER2 cases. For each study patient, formalin-fixed paraffin-embedded (FFPE) tumor samples were retrieved from Institutional Pathology archives. Stromal TILs were assessed according to consensus guidelines^{7 (link),30 (link)} by one investigator (BF) who was blinded to clinical data. AR expression was measured by IHC, using an anti-androgen receptor (Clone SP107; Ventana Medical Systems Inc, pre-diluted) and the percentage of AR-positive nuclei was quantified. A cutoff of 10% or greater was used to define an AR-positive tumor^{31 (link)}. The IHC threshold (= 0 + or < 10% or ≥ 1 + and ≥ 10%) was used for AR.

Immunohistochemistry for ER and PR was carried out on MCF7, T47D, BT474, and SKBR3 cells at baseline and treated with E₂ (10^&8mol L^&1) and TAM (10^&6mol L^&1) for 24, 48, and 96 h. At each time point, cells were harvested, formalin-fixed, and paraffin-embedded according to standard procedures. Sections of the representative cell block were cut at 3 &m and mounted on electrostatically charged slides. Immunohistochemistry was performed using an automated immunostainer (Ventana BenchMark XT AutoStainer; Ventana Medical Systems, Tucson, AZ, USA) with antibodies against ER (Clone SP1, prediluted, Ventana) and PR (Clone 1A6, 1:50 diluted; Leica Biosystems). Positive and negative controls were included for each immunohistochemical run. IHC slides were scanned by using the Aperio system (ScanScope CS System, Vista, CA, USA) for automated counting. To ensure the reliability of the automatic assessment, stainings were reviewed by two pathologists (A S and C M).

Tumor core biopsies were performed before NAC therapy commenced. ER, PgR, Ki-67 and HER2 were assessed by IHC. IHC staining for ER (Confirm anti-ER, rabbit monoclonal primary antibody; clone SP1, Ventana Medical Systems), PR (Confirm anti-PR, rabbit monoclonal primary antibody; clone 1E2; Ventana Medical Systems), ki-67 (mouse monoclonal primary antibody; clone MIB1; Origene), and HER2 (anti-HER2/neu, rabbit monoclonal primary antibody; clone 4B5; Ventana) was performed.
The percentage and intensity of nuclear staining with ER and PR were estimated, and nuclear staining of ≥ 1% of the invasive tumor nuclei was interpreted as positive, in accordance with 2010 ASCO/CAP guidelines. HER2 positivity refers to cases with an IHC score of 3 + or fluorescence in situ hybridization (FISH) amplification (by 2013 ASCO/CAP criteria) for IHC scores of 2+. Patients were separated into three groups for the purposes of our analysis according to ER expression level: ER-negative, ER low positive (ER staining 1-10%), and ER > 10% positive (ER staining > 10%). The ER expression status on the surgical specimen was recorded. The assessments were performed locally by two experienced pathologists.