The D-BIOMARK clinical trial has revealed that denosumab, a monoclonal antibody targeting RANKL, demonstrates immunomodulatory effects in patients with HER2-negative early breast cancer, potentially expanding its therapeutic applications beyond bone health.
The randomized window-of-opportunity trial enrolled 58 evaluable patients with early-stage HER2-negative breast cancer who were candidates for tumor excision as their first therapeutic approach. Patients were randomized 2:1 to either receive two subcutaneous doses of denosumab (120 mg each) administered 7 days apart or no treatment in the control group.
Denosumab's Effect on Tumor Biology
The primary endpoints of the trial were to assess changes in tumor cell proliferation (Ki67) and apoptosis (cleaved caspase-3) between diagnostic biopsy and surgical specimens. Contrary to expectations based on preclinical studies, denosumab did not reduce tumor cell proliferation or increase apoptosis in early breast cancer patients.
"Once the tumor is established, the anti-proliferative and pro-apoptotic effects may be lost," the researchers noted, explaining the discrepancy between preclinical findings and clinical results. This suggests that RANK signaling may be more critical in tumor initiation rather than in established tumors.
Immunomodulatory Effects Observed
Notably, the trial revealed a statistically significant increase in tumor-infiltrating lymphocytes (TILs) in patients treated with denosumab. This increase was particularly evident in luminal B-like tumors and was observed in both pre-menopausal and post-menopausal patients.
While the control group showed a similar trend toward increased TILs, it did not reach statistical significance, suggesting that denosumab may enhance immune infiltration in the tumor microenvironment. The researchers acknowledged that the inflammatory effect induced by the biopsy or differences between biopsy and surgical specimens might have contributed to variations in both groups.
RANK and RANKL Expression Patterns
The study also provided valuable insights into RANK and RANKL expression patterns in breast cancer. Approximately 34.5% of tumors expressed RANK and 30% expressed RANKL in tumor cells at baseline, slightly higher than previously reported in the literature, particularly in luminal tumors.
RANK protein expression in tumor cells correlated with aggressive tumor characteristics, including high Ki67 levels, low estrogen receptor expression, and high histological grade. A novel finding was the association between RANK expression in the tumor stroma and high levels of tumor proliferation, which remained significant even when analyzing only luminal tumors.
Despite these correlations, baseline RANK or RANKL expression did not predict denosumab-driven changes in TILs, suggesting that other factors may influence the immunomodulatory response to denosumab.
Systemic Effects and Safety Profile
The blockade of the RANK-RANKL pathway was confirmed by a significant drop in serum free RANKL levels in the experimental group, while no changes were observed in the control group. However, the serum levels of bone resorption markers did not change significantly, possibly due to technical limitations or the short interval between treatment and assessment.
Denosumab demonstrated an acceptable safety profile, with the most common adverse events being grade 1 or 2 bone pain (27.03%), grade 1 asthenia (10.81%), and grade 1 pain at the infusion site (8.10%). One case of osteonecrosis of the jaw was reported 11 months after treatment in a heavy smoker, classified as grade 3 toxicity.
Implications for Future Research
The findings from D-BIOMARK suggest potential applications for denosumab as an immune response enhancer in breast cancer treatment, particularly in combination with immunotherapy.
"Could denosumab improve these results?" the researchers questioned, referring to the modest pathological complete response rates observed with immunotherapy in luminal B-like tumors. "A booster given by denosumab allowing new combinations of treatments in a tumor microenvironment that would otherwise be cold is of clinical interest."
The researchers noted that preclinical data already show synergy between immune checkpoint inhibitors and inhibitors of RANK signaling in solid tumors, and clinical trials combining these approaches in melanoma and renal cell carcinoma have shown promising results.
Study Limitations and Strengths
The study had several limitations, including a small sample size that limited subgroup analyses, statistical imbalance due to the 2:1 randomization, and tumor heterogeneity, particularly affecting the triple-negative breast cancer cohort.
However, the inclusion of a control group and a larger sample size compared to previous studies allowed the researchers to generate hypotheses regarding denosumab's role as an immune modulator not only in premenopausal women but also in postmenopausal women.
The D-BIOMARK trial provides important clinical validation of preclinical observations and contributes to a better understanding of denosumab's biological effects beyond bone health, potentially paving the way for new therapeutic strategies in breast cancer treatment.
