Study design and patients
We conducted a single-center, prospective cohort study in the context of routine clinical practice rather than a clinical trial, with the aim of optimizing everolimus dosing through pharmacokinetic and pharmacodynamic analysis in patients with mBC. The protocol was approved by the institutional ethics committee of Asahikawa Medical University (#14085). Written informed consent was obtained from each patient prior to participation in the study. Consecutive patients with mBC who started everolimus plus exemestane between June 2014 and December 2019 were prospectively enrolled. The baseline characteristics of the patients are summarized in Table 1.
Everolimus was administered orally at 10 mg once daily in combination with exemestane (25 mg orally once daily). When severe adverse events were present, the everolimus dose was temporarily reduced or interrupted, followed by resumption at a reduced dose. Everolimus treatment was continued until the occurrence of disease progression, unacceptable toxicity, or patient refusal.
In the outpatient setting, we obtained blood samples immediately before each patient’s morning dose to measure individual everolimus Ctrough levels (i.e., C0). Otherwise, blood samples were routinely collected from remnant blood specimens after laboratory testing at each visit. These nontrough blood samples, taken within approximately 4 h after the morning dose, were used to measure individual everolimus peak concentrations (Cpeak). Blood samples for the concentrations C1, C2, C3, and C4 were defined as those obtained at 1 h ± 30 min, 2 h ± 30 min, 3 h ± 30 min, and 4 h ± 30 min post-dose, respectively. Additionally, blood samples taken in the post-absorption phase at 12 ± 2 h and 24 ± 2 h post-dose were used to determine C12 and C24, respectively.
Whole blood samples (150 µL) were deproteinized with 450 µL of methanol/0.2 M ZnSO4 (70/30, v/v) containing the internal standard ascomycin. After vortex and centrifugation, the supernatant was treated with solid-phase extraction using Oasis HLB cartridges (Waters, Tokyo, Japan). The eluent was evaporated to dryness and reconstituted in 200 µL of 50% acetonitrile. After filtration, 50 µL was automatically injected into a liquid chromatography–tandemmass spectrometry (LC–MS/MS) system. Chromatographic separation was performed with a C18 column heated at 65°C and a flow rate of 0.2 mL/min (lower limit of quantification 1 ng/mL, run time 3.5 min). The isocratic mobile phase consisted of a mixture of 5% 10 mM ammonium acetate and 0.1% acetic acid in water and 95% 10 mM ammonium acetate and 0.1% acetic acid in acetonitrile. Analyses were performed in the multiple reaction monitoring mode at ion transitions m/z 976.4 ® 909.3 for everolimus and m/z 810.4 ® 756.7 for ascomycin. Inter- and intra-assay accuracies were ±10% with a precision (coefficient of variation) below 5%.
Genomic DNA was extracted from the peripheral blood of patients using NucleoSpin Blood QuickPure (Takara Bio, Kusatsu, Japan). Based on previous findings regarding pharmacogenetic determinants associated with everolimus metabolism and disposition, we examined CYP3A4*22, CYP3A5*3, and ABCG2 421C>A polymorphisms [13,17,22]. Genotyping was performed using TaqMan SNP genotyping assays (Thermo Fisher Scientific, Tokyo, Japan).
mTOR activity in PBMCs
PBMCs were isolated from whole blood (approximately 2 mL) by Ficoll-Hypaque density gradient centrifugation. Cell counting and viability assessment were performed using the TC20 counter (Bio-Rad, Tokyo, Japan). PBMCs with viability exceeding 90% were used for the subsequent mTOR assay. After centrifugation at 400 x g for 2 min at 4°C, the supernatant was discarded and the cell pellets were resuspended in 100 µL of ice-cold hypotonic lysis buffer containing protease and phosphatase inhibitor cocktails (Nacalai, Kyoto, Japan), and then stored at –80°C until analysis.
The mTOR activity in the extracts of PBMCs was directly measured using the Kinase-linked immunosorbent assay (K-LISA) mTOR activity kit (CBA055, Calbiochem, USA) by assessing phosphorylation of the specific mTOR substrate p70S6 kinase at Thr389, according to the manufacturer’s protocol and a previous study . A recombinant human mTOR enzyme (1362-end) with a specific activity of 186 U/mg (Lot# 2052551-D, Millipore, UK) was used for calibration (standard series, 0–250 ng). The protein concentration of each lysate was determined using the Coomassie Brilliant Blue R-250 Staining Solution (Bio-Rad, Tokyo, Japan) for normalization of mTOR activity. Finally, the mTOR activity in PBMCs was calculated relative to the baseline activity before the start of therapy in each patient.
The efficacy endpoints were progression-free survival (PFS) and overall survival (OS). For safety assessment, all adverse events were graded according to the Common Terminology Criteria for Adverse Events version 4.03. The cumulative incidence of dose-limiting toxicities (DLTs) leading to treatment discontinuation and dose interruption/reduction was estimated by adjusting for competing risks (e.g., death or treatment discontinuation due to disease progression) using the Fine and Gray model . The data cutoff date was March 31, 2020.
The statistical significance of differences in non-parametric values between two groups was analyzed with the Mann–Whitney U test. A receiver operating characteristic (ROC) curve was constructed, and the area under the ROC curve (AUCROC) was calculated to estimate the optimal cutoff value of everolimus Ctrough for predicting development of DLTs. The median PFS and OS were estimated using the Kaplan–Meier method, and the difference between two groups was examined using the Gehan–Breslow–Wilcoxon test. A two-sided P < 0.05 was considered statistically significant. All statistical analyses were performed using STATA software, version 16 (StataCorp LLC, Texas, USA).