Study Design and Conduct

This open label Phase I study was conducted in three cancer centers in Australia with approvals from all relevant institutional Human Research Ethics Committees. (ACT ETH.10.16.218) All participating patients signed an informed consent document in accordance with the Declaration of Helsinki. The trial was registered on the Australia and New Zealand, and American Clinical Trials Registers (ACTRN12617000943347; ClinicalTrials.Gov NCT03505528) and was assigned a UTN of U1111-1197-5518. The study was conducted in patients with locally advanced inoperable or mBC to assess the safety/tolerability, pharmacokinetics, and antitumor activity of the phenelzine/nab-paclitaxel combination therapy over three 28-day cycles.

The study was designed to have 5 cohorts – an initial sentinel cohort of phenelzine titrated from 15 to 45 mg daily, followed by cohorts of 45, 60, 75 and 90mg phenelzine total daily dose (administered in divided equal aliquots three times per day) ( Figure 1 ). Phenelzine has a well-established safety profile for the treatment of depression with FDA recommended starting dose being 15mg three times daily. Therefore, 15mg once daily was chosen as a starting dose in combination with nab-paclitaxel held at a constant dose of 100mg/m². Phenelzine treatment commenced in cycle 1, (day 2 to day 84) and dose was escalated in the absence of any dose limiting toxicity (DLT). Dose escalation of phenelzine followed the Cumulative Cohort Design (CCD) of Ivanova et al. (15), using a target toxicity level (probability of a DLT at MTD of 0.3 (15). Dose-escalation decisions were agreed upon by the Investigators and the study Sponsor based on an assessment of DLTs, adverse events (AEs), and laboratory data during a DLT window of day 2 to day 56. When the dose of phenelzine was more than 45mg/day, treatment was commenced at 45mg/day and escalated to the target dose with weekly 15mg increments. Dose reductions were instituted when a patient experienced grade 3 or 4 adverse event or a DLT. A maximum of one dose reduction was allowed for nab-paclitaxel and phenelzine after cycle 2. Patients who were benefiting from treatment were allowed to continue study treatments until they experienced disease progression or unacceptable toxicity.

Open in a separate window

Figure 1

Study design.

Treatment

The study comprised a maximum of 3 x 28 day cycles of combination nab-paclitaxel (Abraxane, Specialised Therapeutics) and phenelzine (Nardil, Link Medical Products Pty Ltd). Patients received nab-paclitaxel 100mg/m² as an intravenous infusion over a period of 30 mins once a week for three consecutive weeks, with one week break before commencing the next cycle. Phenelzine treatment commenced on Day 2 of Cycle 1 and continued through to the conclusion of the study. Dose modifications or delays of nab-paclitaxel chemotherapy were executed according to standard clinical practice. The highest planned phenelzine dose was 90mg daily (30mg tds) corresponding to the maximum recommended dose for treatment of depression (16).

Purpose and Assessments

The primary objective was to determine the safety and tolerability of the phenelzine and nab-paclitaxel combination and to define the MTD and OBD. The secondary objectives included an assessment of antitumor activity and exploration of relationships between phenelzine dose and CTC/CSC burden, and dynamic real time assessment of the immune response and various cell surface markers associated with epithelial to mesenchymal transition (EMT). CTCs were enriched from liquid biopsies after CD45 depletion (15162, Stemcell Technologies) and removal of red blood cells as previously described (6, 10). CD45-negative enriched cells were stained with different antibodies against proteins related to EMT/CSC and sorted using DEPArray (17). CTC enrichment and enumeration was conducted at the beginning of each cycle (every 28 days) and at the end of study. The OBD was defined as the dose at which optimal LSD1 inhibition and CTC depletion was achieved.

Safety was assessed by physical examination, ECG, clinical laboratory assessments (hematology, biochemistry) for all patients entering the trial and receiving at least one dose of phenelzine. Any AE that occurred after start of study treatment or worsened during study treatment was defined as a treatment-emergent adverse event (TEAE). The severity of adverse events was graded using categories of mild, moderate and severe with reference to CTCAE v4.03 and DLTs were assessed until the end of cycle 3. DLTs were prospectively defined to included grade 3 febrile neutropenia, > grade 2 peripheral neuropathy and any serious adverse events (SAE). ( Supplementary Table 2 ). The MTD was defined as the dose of phenelzine at which 30% of patients experienced a DLT. Measures of antitumor effect were, objective response rate, complete response [CR], partial response [PR], disease control rate [CR+PR+stable disease/SD], progression-free survival (PFS) and overall survival (OS). Response to treatment was assessed using Response Evaluation Criteria in Solid Tumors (RECIST) Version 1.1 (18).

Immunofluorescence Staining and Microscopy

Immunofluorescence imaging and analysis was carried out using previously established and optimized protocols (6, 10). Cells were stained with appropriate primary and secondary antibodies. Protein targets were localized by confocal laser scanning microscopy. Single 0.5 μm sections were obtained using a Leica DMI8 microscope running LAX software using a 100x oil immersion lens. The final image was obtained by averaging four sequential images of the same section. Digital images were analyzed using ImageJ-Fiji software (ImageJ, NIH, Bethesda, MD) to determine either the nuclear, cytoplasmic or whole cell fluorescence. The Two-Way ANOVA test (GraphPad Prism, GraphPad 178 Software, San Diego, CA) was used to determine significant differences between datasets.

Immunofluorescence Analysis: Phenotypic Score

Before the study commenced, it was hypothesized that treatment with nab-paclitaxel and phenelzine would result in a phenotypic change in CTCs. That change was hypothesized to be associated with decreased expression of the markers CSV, EGFR, FOXQ1, LSD1, PD-L1 and SNAI1, and increased expression of the markers H3K9me2 and H3K4me2. The CSV and PD-L1 were measured in cytoplasm. For each cellular localisation, the score consisted of the difference between the average of the markers hypothesised to decrease, and the average of the markers hypothesised to increase. The phenotypic score was plotted against visit and dose, and analysed as above using a mixed effect model with fixed effects for visit and dose and random patient effect ( Box 1 ).

The marker values in the expressions shown above represent the mean of all CTCs for each patient and visit. Scores for each component were plotted against visit (coded numerically- Box 1 ) after treatment, by phenelzine target.

Dose Escalation and Safety

AEs were recorded from the time of the first study dose to the End of Study Visit with a total of 40 TEAEs reported by 8/8 (100%) participants. The most commonly reported events were consistent with the known safety profiles of nab-paclitaxel and phenelzine and included dizziness (including postural dizziness) (n=6), fatigue (n=4), decreased neutrophils (n=3), neuropathy (including peripheral neuropathy) (n=3), diarrhea (n=2) and hallucinations (n=2). All other AEs were single reports. The majority (95%) of TEAEs were either grade 1 or 2. ( Table 2 ) There were no treatment related deaths and no events were reported to be life threatening. Two grade 3 events (2/40, 5%) were reported. The first event was an event of neutropenia in a patient receiving 60mg phenelzine leading to a 1-week delay of nab-paclitaxel treatment. The second event was the only SAE reported during the study and occurred in a patient treated with 45mg phenelzine. This was a prolonged episode of diarrhea resulting in hospitalization (and was classified as a DLT) and resulted in study withdrawal. This patient fully recovered to baseline in 6 days. Another patient experienced lightheadedness/dizziness at the start of Cycle 3 and the phenelzine dose was reduced to 30mg, however the patient subsequently experienced hallucinations and withdrew consent later in cycle 3. These neurological symptoms were considered to be related to phenelzine and the patient fully recovered after ceasing study treatments. One patient in the 60mg cohort experienced increasing confusion and mental fogginess. Phenelzine was ceased for 10 days and then resumed at 45mg for the remainder of the study without further incident.

A reduction in CTC count was seen at both the 45mg and 60mg dose of phenelzine, however CTC count at the commencement of treatment was highly variable and made interpretation difficult. However, phenelzine at 60mg inhibited LSD1 significantly more than at 45mg with an associated increased reduction in mesenchymal phenotype. The study was terminated early at 60mg of phenelzine based on the extent of the observed LSD1 and CTC inhibition, which indicated “proof of biological principle” had been established. In consequence an MTD was not established and, per protocol, 60mg of phenelzine was declared as OBD and recommended phase 2 dose (RP2D).

Efficacy Analysis

Efficacy was included as a secondary objective in this clinical trial via RECIST 1.1 response criteria. Limited efficacy data are available as the majority (75%) of enrolled subjects did not have measurable lesions at baseline, with only two (2/8, 25%) patients assessable for end of study RECIST response. Of these two patients, one had stable disease and one had progressive disease at the end of the study. After a median follow up of 113 weeks, 4/8 (50%) patients were alive with a median PFS of 34 weeks (range 8-88 weeks). Survival for the TNBC patients (n=3) was 57 and 76 weeks, while a 3^rd patient with TNBC was still alive (149 weeks) on further treatment at the time of data cut off of April 2021.

Biomarker Analysis

In this clinical trial the effect of phenelzine treatment on a number of mesenchymal markers including CSV, EGFR, FOXQ1, SNAI1 and PD-L1 were assessed using patient derived circulating tumor cells (CTCs) from liquid biopsies taken at baseline and at 29, 57 and 85 days post start of phenelzine therapy. Overall, treatment with phenelzine displayed a trend of decreased cytoplasmic expression of all mesenchymal markers: CSV, EGFR, FOXQ1, PD-L1 and SNAI1 ( Figure 2 ), however, only CSV and EGFR reduction were statistically significant. Small number of patients in the 60mg cohort may have contributed to this. LSD1 cytoplasmic expression was significantly reduced in both cohorts with a stronger effect observed in the 60mg cohort ( Figure 2 ).

Open in a separate window

Figure 2

Analysis of CTC mesenchymal markers during the study. Analysis of CTC mesenchymal marker and LSD1 in metastatic breast cancer patient derived CTCs. Figure shows differential changes between phenelzine 45mg (blue) and 60mg (Red). CTCs were isolated at baseline, cycle 1 (Day 29), 2 (Day 57) or cycle 3 (Day 85 end of study). (A) Shows changes in mean cytoplasmic intensity of CSV, LSD1s111p (LSD1p), EGFR, FOXQ1, PD-L1 or SNAI1 during the course of trial. (B) Shows changes in nuclear intensity of H3K9me2, H3K4me2, LSD1s111p, EGFR, FOX1Q, or SNAI1. (C) Phenotypic index, measuring the phenotypic change in the patients CTCs was calculated as described in the methods for the cytoplasmic, nuclear and whole cell scores. This score was then plotted for cycle 1 to 3 compared to baseline readings. Significant differences were determined with a two-way ANOVA with multiple comparisons to baseline and are indicated, *<0.05, **<0.01, ***<0.001, ****<0.001, ns, not significant.

Nuclear expression of SNAI1, FOXQ1 and EGFR reduced over time during the course of treatment with phenelzine ( Figure 2 ). Phosphorylated LSD1 (LSD1p) nuclear expression was significantly inhibited in both cohorts with more reduction seen with 60mg of phenelzine. The percentage change of expression relative to baseline was calculated for LSD1p for whole cell, cytoplasmic and nuclear expression as determined by high resolution fluorescent microscopy. Treatment with phenelzine reduced LSD1p expression globally, but most significant inhibition was seen in nuclear compartment compared to cytosolic compartment ( Figure 2 ). Overall, expression was lowest at Day 85 at end of study for both the cytoplasmic and nuclear compartments for LSD1 and the mesenchymal markers.

Consistent with reduction of LSD1 expression, H3K4me2 and H3K9me2 nuclear signals (the target histones for LSD1 mediated demethylation) increased over time. The H3K9me2 increment was significantly higher after treatment with 60mg compared with 45mg ( Figure 2 ). Whole cell fluorescent signal analysis revealed a similar trend to the cytoplasmic or nuclear compartments with strongest effects seen on EGFR expression and LSD1p expression.

One patient with TNBC, was still alive at 149 weeks. This patient has now progressed and has received further systemic therapy. CTC analysis of this patient showed significant downregulation of mesenchymal markers, especially at day 57. The most notable changes for this patient were seen in EGFR throughout the trial, while expression of CSV, FOXQ1 and PD-L1 increased at day 85, however, it should be noted number of CTC obtained at day 85 was quite small ( Figure 3 ).

Open in a separate window

Figure 3

CTC analysis of the patient with triple negative breast cancer with longest survival. The mean cytoplasmic fluorescent intensity (CFI) or the mean nuclear fluorescent intensity (NFI) of mesenchymal markers and PD-L1 were measured in CTC of the patients with triple negative breast cancer with longest survival was. Graphs plot the CFI or NFI mean for Cycle 1 to 3 compared to baseline readings. Significant differences were determined with a two-way ANOVA with multiple comparisons to baseline and are indicated, *<0.05, ***<0.001, ****<0.001, ns, not significant. Example images of CSV, LSD1p and PD-L1 or CSV and EGFR are shown for each time point with the scale bar in orange equal 1 micron.

Phenotypic Score in Metastatic Breast Cancer Patient Derived CTCs

To further probe the change in the mesenchymal signature of the CTCs, a “phenotypic score” ( Box 1 ) was used to calculate the change. This score is based on the fluorescent intensity of the mesenchymal markers (CSV, PD-L1, FOXQ1, EGFR, SNAI1), the drug target, LSD1, and the histone marks (H3K4me2, H3K9me2), ( Figure 2C ). A reduction of phenotypic score indicates a reduction in the overall mesenchymal signature of the CTC. Phenelzine treatment at either the 45 or 60mg doses significantly abrogated the phenotypic score in cytosolic, nuclear and whole cell compartment.

Authors would like to thank members of ACT Pathology in assisting with blood collections and other members of the lab in assisting with sample processing. EpiAxis Therapeutics provided editorial and writing assistance during the development of the manuscript.