Study design and patients
The trial was approved by the institutional review board of the Center for Cancer Research, National Cancer Institute (ClinicalTrials.gov identifier: NCT02484404). Patients eligible for the study had histologically confirmed advanced breast or gynecologic malignancies, measurable by Response Evaluation Criteria in Solid Tumors (RECIST) v1.1. Prior exposure to PARPi or angiogenesis inhibition was eligible but previous treatment with immune checkpoint blockade was not allowed. Patients had to have controlled hypertension with no more than three antihypertensives, and good end-organ function. Germline BRCA mutation status was requested at baseline. All patients provided written informed consent before enrollment.
Eligible patients received all three drugs in a 3 + 3 dose-escalation format as outlined in Table 1. Patients safety was assessed in an ongoing fashion using the Common Terminology Criteria for Adverse Events v4. Response was assessed every two cycles by imaging using RECIST v1.1 criteria. Study treatment was discontinued for progression of disease, intercurrent illness, adverse events (AEs) not recovering to ≤ grade 1 within 14 days, or patient withdrawal of consent.
Definitions of dose-limiting toxicity and maximum tolerated dose
The primary end point was to determine RP2D of the 3-drug combination, defined by the maximum tolerated dose (MTD) or the highest protocol-defined dose in the absence of dose-limiting toxicity (DLT). DLT was defined as grade 3 or 4 non-hematologic and grade 4 hematologic AEs related to study medications occurring during the first cycle (28 days). The MTD was defined as the highest dose level at which one or fewer of six patients experienced a DLT. If the observed AE could be specifically attributed to only one of the drugs, that drug was held while the patient continued to receive the drug not associated with the observed AE. Treatment-related serious AEs occurring within 90 days after the last dose of study drugs were reported.
Patients were asked to take blood pressure (BP) twice-daily and given anti-hypertensive medication(s) if two consecutive readings > 140/90 mmHg. Diarrhea episodes were also recorded in a diary and treated with loperamide per protocol unless associated with immune-mediated colitis.
Pharmacokinetic studies
Plasma samples were collected at 0.5, 1, 2, 4, 8, and 12 h after the first dose(s) of olaparib and cediranib and immediately prior to the second dose of olaparib (approximately 12 h after the first daily dose), on cycle 1 day 1 before durvalumab administration, and again after durvalumab on cycle 2 day 1. Plasma was separated and stored at -80 °C until measurement. The lower limit of quantitation of the assay is 0.5 ng/mL for both olaparib and cediranib using previously reported validated methods [10, 11]. A noncompartmental approach to calculating pharmacokinetics data was employed using Phoenix® WinNonlin v6.4 (Certara Pharsight, Cary, NC).
Archival tissue PD-L1 expression and tumor infiltrating lymphocytes evaluation
PD-L1 expression on archival tissue was a prespecified exploratory end point. PD-L1 labeling of cancer cells and tumor-infiltrating lymphocytes (TILs) was evaluated in archival tissue samples by immunohistochemistry (IHC). The degree of TIL was assessed as a percentage of stromal and intratumoral space occupied by TIL. Unstained slides of the primary tumor sample from all 9 patients were labeled for PD-L1 by IHC with antibody SP263 (monoclonal rabbit; Ventana Medical Systems Inc., Tucson, AZ) on an automated platform [10]. PD-L1 positivity was defined as labeling of ≥1% carcinoma cells or TIL. The PD-L1 labeling by the carcinoma cells and the TIL was assessed and recorded separately.
Pharmacodynamic studies
Immune subsets and functional markers analysis
Peripheral blood mononuclear cells (PBMCs) were collected at baseline (cycle 1 day 1) and on therapy (cycle 1 day 15 and cycle 3 day 1). PBMCs were processed within 2 h and assessed for immune subsets and functional markers using multiparameter flow cytometry as described [12]. CD8+ T cells, CD4+ T cells, Tregs, monocyte subsets and MDSCs were studied. Expression of CTLA-4, T cell immunoglobulin and mucin-domain containing-3 (TIM-3) and PD-1 on CD8+ T cells and Tregs, CD40+ MDSCs, and monocytes HLA-DR and PD-L1 were evaluated as described previously [13]. All analyses were performed using multiparametric flow cytometry (MACSQuant; Miltenyi Biotec, Bergisch Gladbach, Germany), and data were analyzed using FlowJo software v.10.0.7 (FlowJo LLC, Ashland, OR). Flow cytometric data were quantified either as a percentage of a defined cell population, or the median fluorescence intensity (MFI).
Cytokine studies
Plasma samples collected pre- and on treatment were analyzed by ELISA [14]. The pro-inflammatory cytokines IFN γ, IL 10, IL 12, IL 2, IL 6, IL 8 and TNFα were examined.
Statistical analyses
All statistical tests used two-sided significance level 0.05, adjusted for multiple comparisons, using GraphPad Prism software v6.0 (GraphPad Software Inc., La Jolla, CA). Paired t-tests were performed in a parametric manner.