Eligible patients had an advanced solid tumor that is refractory to standard treatment, or for which no standard therapy is available, or the subject refused standard therapy. Other inclusion criteria included: ≥18 years of age, Eastern Cooperative Oncology Group performance status of 0 or 1, adequate organ and marrow function, and measurable disease by Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST 1.1). The first patient enrolled on the study received the first dose of ONC201 on May 2, 2016.

This was a single-arm, single-agent, single-center, Phase I dose escalation and expansion study that evaluated the safety and tolerability of ONC201 in patients with advanced solid tumors. The primary objective was to determine the RP2D, defined as the MTD or the protocol pre-specified MAD of 625 mg, whichever was achieved. Antitumor activity was assessed using RECIST 1.1. Patients with prostate cancer were assessed according to Prostate Cancer Working Group 2 criteria [16]. Secondary objectives included PK, PD, and antitumor activity by RECIST 1.1.

Patients received ONC201 at 375 mg or 625 mg orally once per week, until RECIST 1.1–defined progression occurred. One treatment cycle was defined as 21 days.

Screening and baseline assessments were obtained within 28 days prior to the first dose of ONC201. After baseline evaluation, objective tumor assessments were performed every two cycles (6 weeks ±7 days) until disease progression. Response to treatment was assessed by RECIST 1.1. Blood samples for PK and PD analyses were collected. The safety and tolerability of ONC201 were assessed according to Common Terminology Criteria for Adverse Events (version 4.03). On-treatment biopsies were obtained from two patients: one with prostate cancer and another with endometrioid cancer.

Plasma samples were prepared from blood collected in K₂EDTA tubes at baseline, 30 min, 2, 4, 6, 24, 48, and 168 h following the first two cycles of ONC201 and pre-dose on day 1 of subsequent cycles. PK was analyzed, as previously described [15].

Blood samples for PD analyses were collected at pre-dose, 6 h, 2, 3, 8, and 15 days after ONC201 treatment for cycles 1 and 2, and at pre-dose on day 1 for cycles 3 and beyond. Caspase-cleaved cytokeratin 18 (cCK18) was measured as a biomarker of epithelial cell apoptosis and prolactin was measured as a surrogate biomarker of DRD2 antagonism, as previously described (Diapharma; #P10011) [15]. Immunohistochemistry for CHOP (Proteintech; # 5204–1-AP), DR5 (Novus; NB100–56618), CD56 (BioLegend, Catalog X), Granzyme B (Abcam; Catalog X) and TUNEL (Novus; #NBP2–31164) was carried out per manufacturer’s instructions on slides prepared from formalin-fixed paraffin-embedded tissue. Serum was analyzed for expression of cytokines and effector molecules using LegendPlex assay per manufacturer’s instructions (BioLegend; #746267).

As a Phase I study, results were reported using descriptive statistics. The trial size was driven by the standard 3 + 3 design that has a total enrollment that is dependent on the safety experience. An 11-patient expansion cohort was enrolled to provide more precision around the degree of toxicity and PK profile associated with the RP2D. For assessment of the significance of progression-free survival, a log rank hazard ratio calculation was performed for induction of perforin (GraphPad Prism). For pairwise comparisons, a student’s two-tailed t test was performed (Microsoft Excel).

In total, 20 evaluable patients were enrolled to this trial. During dose escalation, three patients were enrolled in the 375 mg cohort, and six patients were enrolled in the 625 mg cohort. An additional 11 patients were enrolled in the dose expansion cohort who also received 625 mg of ONC201. All patients received at least three doses of ONC201 and thus completed the DLT window. Baseline demographic and clinical characteristics were typical of an adult unselected advanced solid tumor population (Table 1). All patients had prior systemic therapy (predominantly chemotherapy), radiotherapy, and surgery. Tumor types included eight (40%) prostate cancer patients, five (25%) colon cancer patients, four (20%) endometrial cancer patients and three (15%) glioblastoma patients.Tab1

Demographics and clinical characteristics of patients treated with ONC201 who were evaluable for safety and efficacy

No DLT, treatment discontinuation, or dose modifications due to drug-related toxicity occurred in any cohort that was attributed to the study drug (Additional file 1: Table S1). Eight Grade 1 adverse events (AEs) were attributed as possibly related to the study drug. No drug-related Grade > 1 AEs were reported. This included the safety experience during the DLT window and beyond that continued for > 6 months in five patients and one patient who continued therapy for > 50 weeks.

The PK profile of ONC201 during cycle 1 was consistent with previous results from the once every-three-week schedule (Fig. 1) [15]. Expected PK parameters were achieved and include a 4.3 μg/mL Cmax, 34.3 h*μg/mL AUC, and 9.4 h half-life. Comparisons of the PK profile from the first day of cycle 1 (after the first dose of ONC201) and cycle 2 (after the fourth dose of ONC201) revealed similar profiles with no significant differences in PK parameters (Fig. 1b and Additional file 1: Table S2). Thus, weekly dosing of ONC201 did not appear to result in systemic accumulation or altered metabolism that altered its previously reported PK profile.Fig1Fig. 1

Pharmacokinetic analysis of ONC201. a ONC201 plasma concentrations following the first dose of cycle 1 and cycle 2. Concentrations are shown as the mean for each dose cohort in dose escalation (375 mg and 625 mg). b Cmax and AUC for 375 mg (n = 3) and 625 mg (n = 17) dose cohorts for cycle 1 and cycle 2. Each error bar indicates SEM

Serum prolactin was evaluated as a surrogate biomarker of DRD2 antagonism [17]. Consistent with the prior Phase I experience, the majority of patients exhibited a > 2-fold induction of prolactin (Fig. 2a). More than 75% of patients had at least a 50% induction. Induction of serum caspase-cleaved cytokeratin 18 as a biomarker of epithelial cell apoptosis was also observed in 65% of the evaluable patients (Fig. 2b and Additional file 1: Figure S1). The magnitude of induction of these pharmacodynamic biomarkers was similar between the two dose schedules (Fig. 2c). There was no obvious association of either of these serum pharmacodynamic biomarkers with systemic exposure to ONC201 (Additional file 1: Figures S3 and S4) or clinical outcomes. Nevertheless, these PD results supported the previous assertion that 625 mg is a biologically active dose of ONC201.Fig2Fig. 2

Pharmacodynamic assays for ONC201. Maximum fold induction of serum prolactin levels (a) and cleaved cytokeratin 18 (b) in patients relative to baseline levels. c Max fold induction of prolactin and cleaved cytokeratin 18 in patients treated at once every three weeks (Q3W) and on the weekly (Q1W) schedule relative to baseline levels. Median prolactin levels: 1.79 and 1.84 for Q3W and Q1W cohorts, respectively. Median cleaved cytokeratin levels: 1.25 and 1.61 for Q3W and Q1W cohorts, respectively. d IHC analyses of CHOP, DR5, and double-stranded DNA breaks (TUNEL) in baseline and ONC201-treated biopsies for an endometrial cancer patient. The biopsy was done 9.8 months after starting ONC201 treatment (7 days after the most recent ONC201 dose). Each error bar indicates SEM

One additional patient, who had metastatic prostate cancer, underwent an on-treatment lymph node biopsy that was compared to archival tumor tissue from the same lymph node. Comparisons revealed increased infiltration of granzyme B+ and CD56+ cells, corroborating preclinical reports of NK cell intratumoral infiltration in response to ONC201 (Fig. 3a-b). Limited tissue availability precluded interrogation of this patient’s on-treatment biopsy for additional intratumoral PD, such as integrated response activation.Fig3Fig. 3

Immunostimulatory activity of ONC201. Immunohistochemical analysis of CD56+ (a) and granzyme B+ (b) cells in archival and post-ONC201 biopsy tumor tissue of an enzalutamide-refractory prostate cancer patient. Positive staining is depicted in gray color (P < 0.05). * denotes P < 0.05 by student’s two-tailed t test comparing the on-treatment tissue staining to archival tissue staining. c Heat map depicting maximum fold induction relative to baseline of immune cytokines and effector molecules in patients with PFS ≥ 12 weeks versus PFS < 12 weeks. * denotes P < 0.05 by student’s two-tailed t test comparing the on-treatment tissue staining to archival tissue staining. d Maximum fold change over baseline of immune cytokines and effector molecules in patients with PFS > =12 weeks by RECIST who received ONC201 at various dose levels once every three weeks or once weekly. Each error bar indicates SEM. * denotes P < 0.05 by student’s two-tailed t test comparing the maximum values to the baseline value. e PFS in patients who had at least a 50% induction in perforin following ONC201 once every three weeks or once weekly (P = 0.0078; HR 0.3211)

A disease-control rate of 42.9% was achieved amongst all patients. Prolonged stable disease for > 6 months by RECIST criteria was observed in five (23.8%) patients (Fig. 4a). Similar to the prior Phase I experience, regressions in individual metastatic lesions were noted in prostate and endometrial cancer patients. One heavily pre-treated endometrioid cancer patient remained progression-free on treatment for > 70 weeks. Four enzalutamide-resistant prostate cancer patients had PFS > 30 weeks. One prostate cancer patient experienced significant shrinkage (> 20%) by RECIST criteria in primary tumor and bone metastasis after two doses (625 mg) of ONC201 (Fig. 4b). Prostate cancer patients who exhibited prolonged stable disease had stabilized or modest increases in PSA over time, unlike patients who progressed rapidly (Additional file 1: Figure S6).Fig4Fig. 4

Clinical outcome of ONC201-treated patients. a Swimmer plot of ONC201-treated patients showing progression-free survival, as defined by RECIST version 1.1. Each bar represents one patient on the study who has been treated on a weekly schedule with ONC201. Arrowhead indicates that the patient was still on treatment at the time of writing this manuscript. b Primary tumor and bone metastasis measurements from a patient with prostate cancer at baseline and after two doses of weekly 625 mg ONC201