Herceptin

Two HER2⁺ cell lines SKBR3 and BT474 obtained from the American Type Culture Collection (ATCC) were used to investigate whether these four miRNAs contribute to trastuzumab sensitivity. Cells were maintained in RPMI 1640 with 10% fetal bovine serum (FBS). Transfection of the cells with miRNA mimics or miRNA ASOs (Genepharma, Supplementary Table. 7 and 8) was performed using Lipofectamine 3000 (Invitrogen) as we previously described^{56 (link)}. Trastuzumab (Herceptin) was obtained from Genentech and dissolved in sterile water and added to medium at 10 μg ml⁻¹ for 3 days. We performed cell proliferation/apoptosis assay using the CellTiter 96 AQueous Cell Proliferation Assay Kit (Promega) and calculated the percentage of inhibition of cell proliferation as [1−(treated cells/untreated cells)] × 100%. The cell lines were authenticated by short tandem repeat profiling and tested to exclude the mycoplasma contamination by PCR.

Full details of this randomized, double-blind, active-controlled, phase III study (NCT02162667) have been published [10 (link), 11 (link)]. Patients were recruited from 112 centers in 23 countries. Patients received neoadjuvant treatment with eight 3-week cycles of CT-P6 (Herzuma^®; Celltrion, Inc., Incheon, Republic of Korea) or trastuzumab (Herceptin^®; Genentech, San Francisco, CA, USA), both administered at a loading dose of 8 mg/kg on day 1 of cycle 1 and then at 6 mg/kg on day 1 of cycles 2–8, with docetaxel and fluorouracil, epirubicin, and cyclophosphamide, followed by surgery (Fig. S1 in Online Resource 1). Patients then received ≤ 10 cycles of adjuvant CT-P6 or trastuzumab (6 mg/kg administered every 3 weeks, per original randomization) before entering a post-treatment follow-up period, which extended until 3 years from the day of enrollment of the last patient.
Eligible patients were women aged ≥ 18 years with histologically confirmed, newly diagnosed HER2-positive breast cancer of clinical stage I–IIIa per American Joint Committee on Cancer Breast Cancer Staging, Seventh Edition [10 (link)]. Patients were required to have a left ventricular ejection fraction (LVEF) of ≥ 55% at baseline [10 (link)]. Key exclusion criteria included bilateral breast cancer, prior breast cancer treatment, and prior anthracycline treatment [10 (link)].

The recombinant humanised monoclonal HER2 antibody trastuzumab (a concentration of 15 µg/mL was selected as indicated elsewhere [24 (link)]) (Herceptin, Genentech, San Francisco, CA, United States) was supplied by the pharmacy of our hospital; BEZ235 (S1009), everolimus (S1120) and TAK-228 (S2811) were obtained from Selleckchem (Selleckchem Spain, Madrid, Spain).

BT474 was cultured in DMEM supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic under a 5% CO2 environment. BT474⁻ PTEN⁻ LTT cells, which were induced by shRNA knockdown of PTEN and long term treatment of Trastuzumab, were maintained in the same media as the parental cell line. Sulforaphane was obtained from Quality Phytochemicals LLC (New Jersey, USA) and diluted in DMSO (<0.1%) for in vitro studies or 0.9% saline in vivo. Docetaxel (Hospira) and Trastuzumab (Herceptin, Genentech) were obtained through the University of Michigan Cancer Center Pharmacy.

This randomised, double-blind, parallel group, active-controlled phase 3 study (ClinicalTrials.gov identifier: NCT02162667) recruited patients from 112 centres in 23 countries [7 (link)]. Patients were randomised (1:1) using a computer-generated randomisation schedule and entered the neoadjuvant treatment phase consisting of eight 3-week cycles of CT-P6 (Herzuma^®; CELLTRION Inc., Incheon, South Korea) or trastuzumab (Herceptin^®; Genentech, San Francisco, CA, USA) at a loading dose of 8 mg/kg followed by 6 mg/kg for cycles 2–8, with docetaxel and fluorouracil, epirubicin and cyclophosphamide (FEC) as shown in Figure S1 (see Online Resource 3). Following surgery, patients received 6 mg/kg of adjuvant CT-P6 or trastuzumab (per original randomisation [7 (link)]), administered as a 90-min intravenous infusion every 3 weeks until ≤ 1 year from the first neoadjuvant dose (excluding the non-treatment period around surgery), or ≤ 10 cycles post-surgery. Patients received radiotherapy and/or hormonal therapy during the adjuvant period at the investigator’s discretion. A post-treatment follow-up period continues for up to 3 years from the date of enrolment of the last patient.

All reagents used in cell culture experiments were purchased from Gibco BRL (Grand Island, NY, USA) except when noted. All other reagents were purchased from Sigma-Aldrich (Darmstadt, Germany) except where otherwise noted. Anti-HER2 sdAb 2Rs15d and non-targeting sdAb R3B23 were generated as described previously [22 (link),57 (link)]. Trastuzumab (Herceptin^®, Genentech, San Francisco, CA, USA) was used as stated in the experiments.