Combined oral low-dose cyclophosphamide endocrine therapy may improve clinical response among patients with metastatic breast cancer via Tregs in TLSs

Abstract

Despite limited research on refractory and/or endocrine therapy failure in elderly metastatic breast cancer (MBC) patients, a prior study showed that low-dose oral cyclophosphamide (CY) can improve the overall survival rate of MBC patients, possibly through the immunoregulation of regulatory T cells (Tregs). We preliminarily investigated the combination of endocrine therapy (ET) with oral low-dose CY as salvage therapy in elderly patients via peripheral blood regulatory T-cell analyses. In addition, we evaluated the associations of tumor tertiary lymphoid structures (TLSs) with therapeutic outcomes. HR+/HER2- advanced breast cancer patients who received low-dose CY combined with ET or ET only from April 2015 to August 2021 were enrolled in this retrospective study. The primary outcome was the clinical control rate (CCR), and the secondary outcome was progression-free survival (PFS). Circulating T lymphocyte subpopulations represented by Tregs were monitored during treatment by flow cytometry methods. TLSs wereconfirmed by hematoxylin-eosin staining of pretreatment specimens, and CD3, CD4, and Foxp3 were detected using Opal multicolor immunofluorescence. A total of 85 patients who received CY + ET and 50 patients who received ET only were enrolled, the percentage of patients who received CCR was 73% (62/85) vs. 70% (45/50), and the objective response rate (ORR) was 28% (24/85) vs. 24% (12/50). No deaths occurred during the study period. The mean PFS time was 13 vs. 11 months (P = 0.03). In the CY + ET group, decreases in CD4+/CD25+/Foxp3+ T cells (P < 0.001) were favorable for both clinical control and prolonged PFS (P < 0.001). Compared with patients without TLSs, those with TLSs were more likely to have better clinical control and PFS (mean time = 6 months), and a greater number of Treg cells during TLS pretreatment correlated with longer PFS (P = 0.043). Oral low-dose CY combined with standard ET exerts immunological effects by decreasing Treg levels to achieve improved clinical responses. Moreover, patients with TLSs might benefit more from such therapy than those without TLSs, and a high Treg cell count in TLSs before treatment predicts better therapeutic efficacy.

Keywords: Cyclophosphamide; Low dose; Metastatic breast cancer; Regulatory T cell; Tertiary lymphoid structures; Tumor microenvironment.

Figure 1

Prognosis of HR+ breast cancer patients after low-dose CY + ET treatment. (A) Disease control in patients after low-dose CY + ET treatment. (B) PFS of patients after low-dose CY + ET treatment. (C) Patients with reduced peripheral blood Treg cell counts after low-dose CY + ET treatment have a better prognosis. (D) Patients with increased CD8+CD28+ T-cell counts in peripheral blood after low-dose CY + ET treatment had a better prognosis.

Figure 2

Changes in the T lymphocyte subset and plasma cytokines in peripheral blood after low-dose CY + ET treatment. (A) Changes in peripheral blood lymphoid subgroups before and after low-dose CY + ET treatment. (B) Changes in peripheral blood cytokines before and after low-dose CY + ET treatment.

Figure 3

The presence of TLSs and the number of Tregs in TLSs before treatment affect prognosis. (A) Patients who received TLS pretreatment had a more favorable prognosis. (B) H&E staining was used to observe TLSs. (C) The number of TLSs in patients with different prognoses. (D) The presence of TLSs significantly differed in patients with different prognoses. (E) The presence of Treg cells in TLSs and peripheral blood was positively correlated. (F) The number of Tregs in TLSs before treatment affected prognosis. (G) The difference in the number of Treg cells among TLSs was evaluated by multicolor immunofluorescence staining. (H) Patients with higher numbers of Tregs in TLSs had longer PFS after low-dose CY + ET treatment.