Abstract P2-08-10: First results of the randomized window of opportunity clinical trial D-Biomark: Immunomodulatory effect of denosumab in early breast cancer

Abstract Background: Most breast cancers (BC) exhibit low immune infiltration and are unresponsive to immunotherapy. Inhibitors of the Receptor Activator of NFkB (RANK) pathway, such as denosumab, used for the treatment of bone metastasis, have been shown to prevent BC, reducing tumor cell proliferation and survival. Our recent data supports that RANK pathway inhibition in BC cells enhances anti-tumor immune response. However, the population of BC patients who may benefit from denosumab remains to be identified. The aim of the study is to evaluate the antiproliferative, proapoptotic and/or immunomodulatory activity of denosumab in early BC and to identify biomarkers of response. Methods: Patients with early stage HER2-negative BC, candidates to tumor excision as first therapeutic approach were included. Patients were randomized 2: 1 to denosumab (two doses of 120mg/Kg subcutaneous denosumab, days 1 and 8 plus oral calcium 1000 mg daily for a month) and control arm (no treatment). Blood and tumor samples were collected at baseline and at surgery (2-4 weeks later). Putative changes in tumor cell proliferation by Ki67 immunohistochemistry (IHC), survival by Cleaved caspase-3 IHC and tumor infiltrating lymphocytes (TILs) quantified in H&E were evaluated between initial biopsy and surgery. RANK and RANKL expression will be analysed by IHC and the infiltrating immune populations will be characterized by specific antibodies (CD3, CD4, CD8, CD20, FoxP3, CD68, PDL1, PD1). We will analyse denosumab driven gene expression changes in tumor samples and tools such as CIBERSORT will be used to characterize the immune infiltrate. To assess the increase in TILs and the variation in Ki 67 and Cleaved caspase-3, the T-test for paired samples was used. A value of p less than 0.05 was defined as statistically significant. Result: We present results from the initial 45 patients enrolled out of 60, 31 cases in the experimental arm and 13 in the control arm. Mean age was 55,97 (range 37-88) years. 38 women with luminal breast cancer were analysed and 7 cases of triple negative BC (5 in experimental arm and 2 in control arm). Clinical and tumor characteristics were well balanced between both groups. No relevant toxicities were reported. There was no reduction of Ki67 in either of the two arms and no changes in Cleaved caspase-3 were observed. Interestingly, a statistically significant increase in TILs was observed in the denosumab treated group (p=0.0092, Paired t test) but not in the control group (p=0.68). 29.03% of patients treated with denosumab showed a ≥10% increase in TILs vs 7.14% in the control group (p=0.13). Denosumab was associated with an effective systemic inhibition of RANKL. No relationship was found between serum RANKL levels at baseline and response to denosumab. RNAseq analysis and immunophenotyping is still in progress. Conclusion: Short term neoadjuvant denosumab induces an immunomodulatory effect with an increase in stromal TILs in early BC. ClinicalTrials.gov Identifier: NCT03691311 Table 1.ResultsBiopsySurgeryKi 67 (mean frequency) Control24.5729.57Ki 67 (mean frequency) Experimental22.3927.90Cleaved caspase 3 (mean area) Control0.260.58Cleaved caspase 3 (mean area) Experimental0.350.42TILs (mean percentage) Control9.3410.29TILs (mean percentage) Experimental8.2913.54TILs incrementControl p=0.68 vs. Experimental p=0.009 Citation Format: Andrea Vethencourt, Eva M Trinidad, Anna Petit, María T Soler-Monsó, Clara Gómez Aleza, Ander Urruticochea, Amparo García-Tejedor, Anna Gumà Martinez, Veronica Obadia, Silvia Vazquez, Rafael Villanueva, Adela Fernánez, Monica Cejuela, Sabela Recalde Penabad, Agostina Stradella, Miguel Gil-Gil, Sonia Pernas, Eva Gonzalez-Suarez, Catalina Falo. First results of the randomized window of opportunity clinical trial D-Biomark: Immunomodulatory effect of denosumab in early breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-08-10.