Evaluating the efficacy of a priming dose of cyclophosphamide prior to pembrolizumab to treat metastatic triple negative breast cancer

Abstract

Purpose: Triple negative breast cancer (TNBC) is characterized by the presence of immune cells in the tumor microenvironment, however, the response to single-agent immune checkpoint inhibitor (ICI) therapy is modest. Preclinical models have demonstrated that intratumoral regulatory T cells (T_regs) dampen the antitumor response to ICI. We performed a single-arm phase II trial to evaluate the efficacy of a single low dose of cyclophosphamide (Cy) to deplete T_regs administered before initiating pembrolizumab.

Patients and methods: 40 patients with pretreated metastatic TNBC were enrolled. The primary endpoints were progression-free survival (PFS) and change in peripheral blood T_regs after Cy. Secondary endpoints included overall response rate (ORR), duration of response, overall survival, treatment-related adverse events (AEs), and correlative evaluations.

Results: Median PFS was 1.8 months, and the ORR was 21%. T_regs were not significantly decreased after Cy prior to ICI (-3.3%, p=0.19), and increased significantly after the first cycle of therapy (+21% between cycles 1 and 2, p=0.005). Immune-related AEs were similar to historical pembrolizumab monotherapy, and were associated with response to therapy (p=0.02). Patients with pretreatment tumors harboring increased expression of B cell metagene signatures and increased circulating B cell receptor repertoire diversity were associated with clinical response and immune-related toxicity (IRT).

Conclusions: Among patients with heavily pretreated TNBC, Cy prior to pembrolizumab did not significantly deplete T_regs, and in those with decreased numbers there was rapid recovery following therapy. Increased B cell gene expression in baseline samples was associated with clinical response and IRT.

Keywords: breast neoplasms; clinical trials; immunotherapy; phase II as topic; programmed cell death 1 receptor.

Figure 1

Adverse events and efficacy of treatment on peripheral T_regs. (A) Most common toxicities by grade (1–2 or 3–4). (B) Levels of peripheral CD45+/FOXP3+/CD4 +regulatory T cells from blood collected on C1D1, C1D2, and C2D1, and comparison between time points.

Figure 2

Survival outcomes, response rates and immune-related toxicities. Kaplan-Meier plots of (A) progression-free and (B) overall survival of patients in months, with medians and 95% CIs indicated. Vertical red dotted lines in A denote the null (1.9 months) and alternative (2.9 momths) hypotheses. n=40 patients. (C) Swimmer’s plot of patient responses (complete response, partial response, stable disease, or progressive disease) and whether patients experienced immune-related toxicities (yes/no) over time (months). Each bar represents one patient, n=39 patients. The x axis represent time on treatment, starting at initiation of protocol therapy and ending when the patient dies or is censored.

Figure 3

Tumor genomic and immune features. (A, B) Tumor PD-L1 expression was not significantly associated with either clinical benefit (CR+PR+SD; A) or response (CR+PR; B) to therapy by t-test (shown), or by stratifying PD-L1 positive/negative (PD-L1 <1% vs ≥1%, or PD-L1 <10% vs ≥10%; Wilcoxon rank-sum test, not shown). (C) Differential gene expression (α<0.2) in archival tumor samples, by treatment response (CR+PR; bottom rows) vs non-response (SD+PD; top rows). (D) Frequently somatically mutated genes implicated in breast cancer per COSMIC Tier one classification by variant type. Raw tumor mutational burden is noted at the top of each sample column. Treatment response and tumor PAM50 subtype for each sample is listed at bottom of each column. CR, complete response; ns, not significant; PD, progressive disease; PR, partial response; SD, stable disease.

Figure 4

Immune repertoire diversity. (A–D) Univariable Cox proportional hazards models for PFS showing adaptive immune receptor repertoire diversity measures derived from pre-treatment tumor RNA-Seq (A), PBMC-derived amplicon sequencing pre-pembrolizumab (B), and PBMC-derived amplicon sequencing post-pembrolizumab (after at least 2 cycles of pembrolizumab) (C). For readability, TRA chain metrics from pretreatment tumor RNA-Seq are displayed in (D). Bright red indicates measures which were significant after FDR adjustment at α<0.20, dark red at p<0.05. FDR, false discovery rate; PBMC, peripheral blood mononuclear cell; PFS, progression-free survival.