Denosumab as an immune modulator in HER2-negative early breast cancer: results of the window-of-opportunity D-BIOMARK clinical trial.

Denosumab as an immune modulator in HER2-negative early breast cancer: results of the window-of-opportunity D-BIOMARK clinical trial. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Denosumab as an immune modulator in HER2-negative early breast cancer: results of the window-of-opportunity D-BIOMARK clinical trial. Andrea Vethencourt, Eva M. Trinidad, Eduard Dorca, Anna Petit, and 16 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4283385/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 12 May, 2025 Read the published version in Breast Cancer Research → Version 1 posted 9 You are reading this latest preprint version Abstract Background: The RANK pathway has been extensively investigated for its role in bone resorption; however, its significance extends beyond bone metabolism. Preclinical models suggest that inhibition of RANK signaling can prevent mammary tumor development by reducing proliferation and tumor cell survival. Additionally, both preclinical and clinical data support the ability of RANK pathway inhibitors to enhance the anti-tumor immune response. Methods: D-BIOMARK is a prospective, randomized window-of-opportunity clinical trial assessing the biological effects of denosumab, a monoclonal antibody against RANKL, in patients with HER2-negative early breast cancer. The study aims to assess denosumab's impact on breast tumor cell proliferation, apoptosis, and its potential to influence the tumor immune microenvironment. A total of 60 patients were enrolled and randomized 2:1 to receive two doses of single agent denosumab (120 mg one week apart) before surgery or to the control arm (no treatment). Fifty-eight patients were evaluated, 27 pre-menopausal, and 31 post-menopausal women, 48 with luminal tumors and 10 with triple negative breast cancer. Paired tumor samples were collected to compare baseline (core biopsy) and surgical (surgical specimen) time points, as well as serum samples at both time points. Results: Denosumab demonstrated its ability to reduce serum free RANKL levels (experimental p<0.001, control p=0.270). However, a reduction in tumor cell proliferation or cell survival was not observed. A denosumab-driven increase in tumor infiltrating lymphocytes (TILs) was observed (experimental p=0.001, control p=0.060), particularly in the luminal B-like population (experimental p=0.012, control p=0.070) and a similar trend in the TNBC group (experimental p=0.079, control p=0.237). Denosumab led to increased TILs in both pre-menopausal (experimental p=0.048, control p=0.639) and post-menopausal (experimental p=0.041, control p=0.062) women with luminal tumors. RANK protein expression in tumor and stroma was associated with markers of tumor aggressiveness but an increase in TILs was observed in the experimental arm irrespectively of RANK and RANKL expression in tumor or stromal cells. Conclusions: The D-BIOMARK trial highlights the potential of denosumab as an immune-enhancing agent in early HER2-negative breast cancer. Although preoperative denosumab did not reduced tumor proliferation or increased apoptosis, it led to an increase in TILs, particularly in luminal B-like tumors. These findings underscore the importance of further investigation into the multifaceted aspects of the RANK pathway. Trial registration: EudraCT number: 2016-002678-11 registered on June 15, 2018. ClinicalTrials.gov identifier: NCT03691311, retrospectively registered on September 04, 2018. breast cancer RANK RANKL denosumab TILs immune enhancer tumor cell proliferation tumor cell survival HER2-negative Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction The receptor activator of nuclear factor κB, known as RANK, member of tumor necrosis factor receptor (TNFR) superfamily, and its ligand (RANKL) have emerged as potential therapeutic targets in breast cancer (BC) and other solid tumors (1–3). Binding of RANKL to RANK leads to activation of signaling pathways related to tumor proliferation, survival, and inflammation, such as the canonical and non-canonical NFκB, MAPK and PI3K-AKT. Osteoprotegerin (OPG), a natural negative regulator of the RANK pathway acts as a decoy receptor and prevents the binding of RANKL to RANK. The RANK/RANKL/OPG axis has been widely studied as a regulator of bone resorption, leading to the development of denosumab, a highly specific immunoglobulin type IgG2 monoclonal antibody, which binds with high affinity to human RANKL and neutralizes its activity (4). Denosumab is approved for the prevention of skeletal events in patients with bone metastases and for the treatment of unresectable giant cell tumors of bone and osteoporosis (3,5,6). Preclinical data show that RANK signaling regulates mammary gland development and mammary cell fate (7–10). RANKL is the main mediator of the proliferative and pro-tumorigenic role of progesterone in the mammary gland. Pharmacological or genetic inhibition of the pathway prevents or attenuates mammary tumor appearance, reduces cell proliferation in preneoplastic lesions and tumor cell survival in mouse adenocarcinomas (2,11). Blockade of the RANK pathway also reduces the incidence of lung metastases and enhances the differentiation of tumor cells in mouse models (2,11–14). In human mammary adenocarcinomas, RANK protein expression, detected by immunohistochemistry (IHC), is found in 15-20% of estrogen receptor (ER)-positive tumors and in 40% of ER-negative breast adenocarcinomas. RANK protein expression in tumor cells associates with an aggressive tumor phenotype, including hormone receptor-negative tumors, high histological grade and high proliferative index (15–19). Recent findings underscore that RANK protein expression in tumor cells serves as an independent maker of adverse prognosis in post-menopausal patients and ER-negative BC (19). RANK signaling plays a crucial role in regulating the delicate balance between tolerance and immunity (20). RANK is predominantly expressed by myeloid cells such as dendritic cells and macrophages while RANKL expression has been observed on T cells in both the tumor microenvironment (TME) and locoregional lymph nodes. Despite strong preclinical evidence, the therapeutic benefit of denosumab in BC patients beyond its bone-related effects is unclear. In the adjuvant setting, the phase III ABCSG-18 clinical trials clinical trial demonstrated that administering denosumab at 60 mg every 6 months for 5 years, alongside an aromatase inhibitor, not only delayed bone fractures and improved mineral bone density but also increased 8-year disease-free survival (DFS) from 77.5% to 80.6%, with a hazard ratio (HR) of 0.82 (95% confidence interval CI=0.69-1.98), p=0.02. In 2022, further analysis confirmed these benefits, including progression-free survival, bone metastasis-free survival, and overall survival, leading to a recommendation for routine use of denosumab as adjuvant therapy in post-menopausal women with hormone receptor-positive BC (21–23). In contrast, in the D-CARE clinical trial, with adjuvant denosumab at a dose of 120 mg every 4 weeks during 6 months and then every 12 weeks up to five years, did not show changes in bone metastasis-free survival (HR 0.97 (95% IC 0.82-1.14), p=0.70) and reported a similar 5-year DFS between the two groups (HR: 1.04 (95% IC 0.91-1.19), p=0.57) (24). Both studies involved different populations and yielded discordant results. In the neoadjuvant context in the GeparX study, the addition of denosumab to neoadjuvant treatment did not increase the pathological complete response (pCR) rate in early BC (25–28). The D-BEYOND, a prospective single-arm window-of-opportunity trial, evaluated the biological effect of denosumab in pre-menopausal women diagnosed with early BC. It included 27 patients, only one was triple negative breast cancer (TNBC). All patients received two subcutaneous injections of denosumab (120 mg/dose) separated by one week prior to breast surgery. The baseline biopsies were compared with the surgical sample. The study did not meet its primary aims: a reduction in cell proliferation nor an increase in cell apoptosis was observed. However, a brief course of denosumab induced an increase in the inflammatory infiltrate measured by tumor-infiltrating lymphocytes (TILs), especially CD8+ T lymphocytes (29). Here, we present results from the D-BIOMARK trial (NCT03691311), a window-of-opportunity study designed to assess the biological activity of single-agent denosumab in patients with primary operable HER2-negative BC. 2. Materials And Methods 2.1 Trial design and patients D-BIOMARK (NCT03691311) is a prospective, single institution, randomized window-of-opportunity clinical trial evaluating the biological effects of single-agent denosumab in treatment-naive patients with early HER2-negative BC who were candidates for tumor excision as the first therapeutic approach. Exclusion criteria included osteonecrosis of the jaw or risk of developing it, other active malignancies, hypocalcemia, or known hypersensitivity to denosumab. Randomization was stratified by menopausal status and ER-status (ER+ vs triple negative). Post-menopausal was defined clinically as more than 1 year with amenorrhea, or ≥ 60 years old (30). Early-stage breast cancer is defined as stage I, stage IIA, stage IIB, and stage IIIA breast cancers, which denote cancer that has not spread beyond the breast or the axillary lymph nodes (31). Written informed consent was obtained from all patients prior to any procedure within the study. Patients were randomized 2:1 to the experimental or control arm; the experimental group received two subcutaneous doses of denosumab (120 mg each) administered 7 days apart, while the control group received no treatment. Screening visits were conducted for all participants. In both arms, a biopsy was performed at the time of diagnosis (referred to as "biopsy"), with serum collected also at baseline (referred to as "serum A"). Furthermore, a secondary biopsy of the surgical specimen was performed two to four weeks after enrollment, at the time of the surgical excision of the breast tumor (referred to as "surgery"), accompanied by a second blood sample collection (referred to as "serum B"). Additional visits and blood analyses were conducted in the experimental arm before each treatment session. Both groups underwent two follow-up visits for safety assessment at one month and six months post-surgery (Figure 1). Patients in the experimental group received daily calcium supplements (≥ 500 mg elemental calcium) and vitamin D (≥ 400 IU) for one month following the first dose to prevent hypocalcemia. The study protocol was approved by the institutional ethics committee (Research Ethics Committee of the Hospital Universitari De Bellvitge) protocol code PR035/21 and conducted in accordance with the ethical standards outlined in the 1964 Declaration of Helsinki. Clinical data and biological characteristics of the tumors were prospectively obtained from medical records and pathology reports, respectively. Adverse events were recorded starting from the day of obtaining signed informed consent and continued until six months following surgery. The safety data were assessed in accordance with the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE v5.0). 2.2 Tumor assessment The evaluation of conventional BC markers, including ER, progesterone receptor (PR), HER2, and Ki67, was performed in the Pathology Department of the Hospital Universitari de Bellvitge. The status of ER and PR was defined according to the guidelines of the American Society of Clinical Oncology and the College of American Pathologists (ASCO-CAP 2010) (32). The histological grade was evaluated following Nottingham classification (33). The surrogate subtypes of BC were defined according to St Gallen 2015 consensus meetings, using IHC substitutes as follows: luminal A-like: ER and/or PR(+), HER2(-), Ki67 < 20%; luminal B-like: ER and/or PR(+), HER2(-), Ki67 ≥ 20; TNBC: ER(-), PR(-), and HER2(-), regardless of the Ki67 score (34). The objectives of this trial were tested by comparing the biopsy at diagnosis (biopsy) with the surgical specimen (surgery). The evaluation of tumor cellularity was assessed in hematoxylin-eosin (H&E) staining tissue sections. For patients with multiple samples, the sample with the highest tumor content was chosen. The percentage of Ki67, cleaved caspase-3 and TILs were independently evaluated by at least two pathologists specialized in BC, blinded to clinical and experimental data. For Ki67, the antibody used was MIB-1, Agilent Dako, the fixation conditions, processing, and results evaluation were performed according to international recommendations (35,36). TILs were evaluated on H&E-stained slides using standardized methodology (37). The anti‐cleaved caspase‐3 was assessed using Asp175, Cell Signaling; 1:200; the quantification of cleaved caspase-3, as an experimental parameter, has no international quantification guidelines; by recommendation of expert pathologists, it was performed using QuPath® bio-image analysis software and a H-score = (% of cells with weak intensity × 1) + (% of cells with moderate staining × 2) + (% of cells with strong staining × 3). The maximum possible H-score is 300, corresponding to 100% of cells with strong (3+) intensity. To evaluate RANK and RANKL, paraffined tissue sections (4 μm) were used. For each patient, representative unstained slides of the tumor were shipped to NeoGenomics Laboratories (California, USA) for IHC staining of RANK (N1H8, Amgen), RL (M366, Amgen), blinded to clinical information. The percentage of stained cells and their intensity (0, negative; 1+, weak; 2+, moderate; and 3+, strong) in the tumor cells were reported by NeoGenomics, and the H-score was calculated using the previous formula. Due to the complexity of RANK and RANKL IHC interpretation and its reading, a double evaluation of H-scores in tumor cells was performed (NeoGenomics and at the laboratory of Dr. Gonzalez-Suarez). In addition, RANK and RANKL H-scores in stroma were evaluated only at the laboratory of Dr. Gonzalez-Suarez (2). 2.3 Serum analysis Serum concentrations of human free RANKL (sRANKL), tartrate-resistant acid phosphatase 5b (TRACP5b), carboxy-terminal collagen crosslinks (CTX), and OPG were quantified utilizing an enzyme-linked immunosorbent assay (ELISA), in accordance with the manufacturer’s guidelines. Progesterone, estradiol, follicle-stimulating hormone (FSH or Follitropin), calcium, albumin, and blood count values were extracted from laboratory reports processed at the Hospital Universitari de Bellvitge laboratory, where the patient's routine tests were performed. 2.4 Statistical analysis The total patient sample size was defined as 60: 40 in the treatment arm and 20 in the control arm. Since there were no prior data on the distribution, mean and standard deviation of the primary endpoint available at the time of the protocol design, a conventional statistical design for sample size calculation was not feasible; therefore, the trial was designed according to Larry V. Rubinstein's suggestions for phase 0 trials (38). For biological purposes, a minimum of 10 TNBC tumors and a minimum of 24 pre-menopausal patients were included. Assignment to each arm was done by stratified block randomization. All analyses were performed using R version 4.1.3 (available at www.r-project.org), GraphPad Prism version 5 and IBM SPSS Statistics version 25 (IBM Corp, Armonk, NY, USA). At baseline (biopsy/serum A) vs. at surgery (surgery/serum B) values were compared using a paired t-test or McNemar test for numeric and binary variables, respectively. Independent samples t-test was used to compare differences between groups, while chi-squared or Fisher exact test, when appropriate, was used for binary variables. To compare baseline variables and possible predictive factors for response to denosumab, the Mann–Whitney U and Fisher’s exact tests were used for continuous and categorical variables, respectively. All correlations were measured using Spearman’s non-parametric rho coefficient. A Logistic regression analysis was performed to define the odds ratio of developing a response variable. All reported p -values were two-tailed. Statistical significance was set at 0.05. 3. Results 3.1 Patient Population A total of 60 patients were enrolled in the study between August 2018 and May 2021. Two patients were excluded. One of them received neoadjuvant letrozole during the SarsCov2 pandemic period due to a delay in the scheduled date of surgery, and other patient only received one dose of denosumab due to withdrawal of consent after the first infusion. One patient who underwent all the procedures but did not receive denosumab due to an administrative error was transferred to the control group. Thus, a total of 37 patients were analyzed in the experimental group and 21 patients in the control group, all 58 patients completed the follow-up period (Figure 2). The analysis was done by protocol. The clinicopathological baseline characteristics of the evaluable patients are shown in Table 1. The mean age of the study population was 56.4 years (range, 37-80 years), with a mean age of 57 years in the experimental arm and 55.4 years in the control arm. The mean time between the first administration of denosumab and surgery was 21 days. Of the total participants, 27 were pre-menopausal women, with 17 (45.9%) in the experimental arm and 10 (47.6%) in the control group, meeting the requirement of including at least 24 pre-menopausal women. The distribution of post-menopausal patients was: 20 (54.1%) in the experimental arm and 11 (52.4%) in the control group. Most patients were classified as IA clinical stage, with 26 cases (70.3%) in theT experimental arm and 17 cases (81%) in the control arm. The median tumor size was 18 mm (range 8-45 mm). In terms of tumor characteristics, the majority of patients had invasive breast cancer without specific features, classified as no special type (NST) or not otherwise specified (NOS) (ductal), 62.07% overall; with a slightly higher percentage in the control arm (71.4% vs. 56.8% in the experimental group), with a lower number of patients with invasive lobular carcinoma in the control arm (14.3 vs. 32.4% in the experimental arm), although these differences were not significant. Most tumors had a histological grade 2 (58.62%), with 7 tumors (12.06%) classified as grade 3; notably, there was a higher percentage of cases with histological grade 1 tumors (35.1%) in the experimental group compared to the control group (19%). The data are consistent with a higher number of cases with low Ki67 (<15) in the experimental group (43.2%) compared to the control group (9.5%). Ki67 was the only parameter that showed significance (p=0.001) when comparing both groups; however, when analyzing the tumors by surrogate molecular subtype, this difference was not significant, although numerically, there were still more cases of luminal A-like tumors in the experimental arm (51.4% vs. 38.1%). The groups were well balanced as no statistically significant differences between experimental and control arms were observed, except for the percentage of patients with tumors with low Ki67 (<15%) (Table 1). Of the total of 58 evaluable cases, 48 were luminal tumors (27 luminal A-like and 21 luminal B-like), while 10 were TNBC. Initially, a higher percentage of TNBC cases was expected. However, most TNBC tumors were selected for neoadjuvant treatment and were therefore excluded from our study. Upon reviewing these 10 TNBC cases, only 5 exhibited typical aggressive characteristics (histological grade 2/3, Ki67 >30%), with 3 of these cases in the control group and 2 in the experimental arm. The remaining cases were low aggressive TNBC, including 3 cases of invasive carcinomas with apocrine differentiation, 2 in the experimental arm and 1 in the control arm: apocrine carcinomas are known to exhibit a more indolent behavior compared to typical TNBCs. Additionally, the experimental group had 1 case of lobular carcinoma and 1 case of carcinoma not otherwise specified (NOS) ductal, with Ki67 of 10% and 5%, respectively. Given these factors, caution should be exercised in drawing conclusions about the triple negative subgroup. 3.2 Safety Data Five patients experienced localized hematomas in the breast following study biopsies, which were classified as grade 1 and did not require drainage or special measures; 2 of these cases were in the control group and 3 were in the experimental group. In the experimental arm, the most common adverse events were grade 1 or 2 bone pain occurring 24 hours after infusion in 10 of 37 patients (27.03%), grade 1 asthenia in 4 patients (10.81%), grade 1 pain at the denosumab infusion site in 3 patients (8.10%), grade 1 chills in 2 patients (5.41%), and grade 2 dental infection in 1 patient (2.70%). There were no reported cases of hypocalcemia. No grade 3 toxicities were reported (see Table S1 in the Supplementary Appendix). Outside the study follow-up period, we observed a long-term event of osteonecrosis of the jaw in a heavy-smoker patient who received denosumab on March 10 th and 17 th , 2021. This patient was initially reported with a dental infection. Symptoms began one month after the last dose of denosumab (reported on April 19, 2021), with discomfort and pain in the jaw, the case was referred to the Maxillofacial Surgery Department. Computed tomography initially did not reveal signs of osteonecrosis, and the case was initially diagnosed as a tooth infection, which improved with oral antibiotic treatment. However, repeated episodes of dental infection in the same location needed specific follow-up. After 11 months, the patient underwent jaw surgery in February 2022, and the pathological report confirmed osteonecrosis of the right quadrant 44-47. This event was considered possibly related to denosumab, although other triggering risk factors such as chronic infection due to long-term smoking should be considered. The osteonecrosis of the jaw was classified as grade 3. At the time of this report, no grade 4 or 5 toxicity has been reported. 3.3 Serum Analysis Denosumab was associated with systemic inhibition of RANKL but not with changes in bone remodeling markers The blockade of the RANK-RANKL pathway was confirmed by the drop in serum of free RANKL (sRANKL), measured by ELISA, in the experimental group as RANKL became bound to denosumab (mean serum A 0,096 pg./L vs serum B vs. 0,000 pg./L p<0.001), while no changes were found in the control group (mean serum A 0,100 pg./L vs. serum B 0,116 pg./L; p=0.270) comparing serum A vs serum B. OPG levels tended to increase in the experimental group (p=0.071), consistent with the reduction of free RANKL. However, the serum levels of the bone resorption markers t artrate-resistant acid phosphatase 5b (TRACP5b) (n=37) and carboxy-terminal collagen crosslinks (CTX) (n=38) did not change in any group (Figure 3 and Supplementary data Figure S1). It is unclear whether the lack of changes is due to technical limitations (limited detection rate) or due to the kinetics of bone resorption. Although no significant alterations were observed in bone resorption markers, a small decrease in serum calcium -not clinically relevant- was reported in the experimental arm (Figure 3), despite the prescribed calcium and vitamin D supplementation. This finding reinforces the efficacy of denosumab in bone remodeling. A correlation analysis was conducted between the different serum markers studied and menopausal status to better understand the biology of the pathway. Follicle-stimulating hormone (FSH) levels were higher in post-menopausal patients, consistent with menopausal physiology (p<0.0001). Levels of free sRANKL did not differ based on menopausal status, while higher levels of OPG were detected in post-menopausal women (p=0.010) at baseline (Supplementary data Figure S2A). Comparable values of the bone markers TRACP5b, and CTX were found at the time of diagnosis (baseline) between pre- and post-menopausal women, although slightly higher levels were found in the post-menopausal group. In pre-menopausal patients, no associations were found between levels of progesterone and sRANKL in serum (p=0.401). Correlation between OPG and TRACP5b values was not significant, unless one sample with high OPG and low TRACP5b was excluded. Finally, a negative correlation between OPG and sRANKL was demonstrated (p=0.0026), consistent with the known interaction between these factors (Supplementary data Figure S2B). 3.4 Tumor assessment´s results Denosumab was not associated with a reduction in tumor cell proliferation or an increase in apoptosis The primary endpoints of the clinical trial were a decrease in tumor cell Ki67 and an increase in apoptosis between biopsy and surgery. Denosumab did not reduce tumor cell proliferation or survival between paired biopsy and surgery samples (Figure 4A-B). The percentage of tumor cells expressing Ki67 increased in both groups (control p=0.035 and experimental p=0.012), which may be attributed to a higher quantification of fields within the surgical specimen (more fields) compared to the core biopsy. The mean Ki67 in the control arm increased from 24.52% at biopsy to 29.19% at surgery. Similarly, in the experimental arm, it increased from 20.86% to 24.81%. This nearly 5-percentage-point increase when comparing surgical samples with baseline biopsies in both groups suggests that these changes were not influenced by denosumab treatment. Indeed, a comparison between the experimental and control groups revealed identical behavior (p=0.928) (Figure 4A). Denosumab did not induce an increase in tumor cell apoptosis, as demonstrated by the assessment of the H-score of cleaved caspase-3 between biopsy and surgery. The comparison between the experimental and control groups revealed notable inter-patient variability. Although a statistically significant difference in apoptosis was observed inter-group (p=0.042), the change in cleaved caspase-3 H-score quantification was less than 1 in both groups. Despite the contrasting trends, the alteration in apoptosis is considered clinically insignificant due to very low H-scores in all cases. The evaluating intra-patient (Paired T-test) showed no changes (control group p=0.060 and experimental p=0.238). Additionally, 3 patients in the experimental group exhibited higher levels of cleaved caspase-3 at baseline (Figure 4B) that may suggest potential deterioration or non-specific staining in some areas. A subgroup analysis was conducted to elucidate if any specific patient group could benefit from denosumab treatment. Patients were divided according to surrogate molecular subtype (Supplementary data Table S2). Patients with luminal A-like (n=27) and luminal B-like (n=21) tumors showed similar trends to the overall population: there was no reduction in Ki67 or increase in Cleaved Caspase-3 in the experimental group and no changes were observed in OPG or TRACP5b. In the TNBC group (n=10), there was an increase in Ki67 in the experimental arm (p=0.025), which was not evident in the control group (p=0.517), but no difference was found in the inter-group comparison (p=0.197). Moreover, it is important to note the small number of TNBC cases, with 6 in the experimental group and 4 in the control group, as well as an imbalance between aggressive tumors, as previously explained (Supplementary data Table S2). A subgroup analysis was also conducted based on menopausal status, excluding cases of triple-negative tumors to avoid biasing the information, as 9 out of 10 TNBC tumors were post-menopausal. Both pre-menopausal (n=26) and post-menopausal (n=22) tumors showed similar trends, with no reduction in proliferation or cell survival, and no other notable findings (Supplementary data Table S3). Denosumab increased tumor infiltrating lymphocytes in early breast cancer, particularly in luminal B-Like tumors and regardless of menopausal status Next, we interrogated the effect of denosumab on tumor immune infiltration and observed an increase in TILs in the surgery sample compared to the initial biopsy in the experimental arm (p=0.001), but not in the control arm (p=0.06). However, both groups showed a similar trend (p=0.789) (Figure 5A-B). Performing a similar analysis based on surrogate molecular subtype, we found that while no changes in TILs were noted in Luminal A tumors (experimental (p=0.144), control (p=0.958)), there was a denosumab-induced elevation in TILs in luminal B-like tumors (p=0.012), with no significant changes in the control group (p=0.070). TILs did not change in TNBC (experimental (p=0.079), control (p=0.237)), although an increased number of representative TNBC tumors is required to reach conclusions in this subtype. Attending to menopausal stage in luminal tumors, we found that both pre-menopausal and postmenopausal patients experienced an increase in TILs in the experimental arm (premenopausal: p=0.048, postmenopausal: p=0.041), but not in the control arm (premenopausal: p=0.639, postmenopausal: p=0.062). In all comparisons the experimental and control arms showed similar trends (Table 2). Furthermore, applying a threshold of a 10% or greater increase in TILs between biopsy and surgery samples, we observed that 9 out of 37 (24.3%) patients in the experimental arm and 5 out of 21 (23.8%) patients in the control arm showed a clinically significant increase in TILs. Tumor and stroma RANK expression was associated with highly proliferative tumors The quantification conducted externally by NeoGenomics and in-house by the laboratory of Dr. Gonzalez-Suarez showed perfect correlation in the analysis of tumor RANK and RANKL protein expression (Supplementary data Figure S3). Given the correlation and the fact that the quantification of H-score for RANK and RANKL in the stroma was only conducted in the laboratory of Dr. Gonzalez-Suarez, all subsequent analyses were carried out using in-house quantification. A total of 55 cases were assessable for tumor RANK and RANKL expression at baseline (Table 3, Figure 6A-B) and 19 tumors (34.54%) exhibited positive baseline expression of RANK, defined as an H-score > 0 (tumor RANK+). Of these positive cases, 14 tumors were randomized to the experimental arm (38.9%), and 5 were assigned to the control group (p=0.526, well balanced). The frequency of RANK+ tumors was comparable between tumors from premenopausal (36%) and postmenopausal patients (33%). Additionally, we compared tumor RANK expression across different molecular subtypes, 28% of luminal A-like, 35% of luminal –B-like and 50% of triple negative tumors exhibited RANK+ tumor cells, a higher frequency than that previously reported, particularly in luminal tumors (18,19). A total of 42.4% of grade 2 and 42.9% of grade 3 tumors were positive for tumor RANK, compared to only 13.3% of grade 1 tumors. Regarding RANKL expression, 17 out of 55 tumors (30.10%) exhibited RANKL expression in tumor cells, with 12 assigned to the experimental group and 5 to the control group (p=0.819, well balanced). The frequency of tumor RANKL expression was similar between pre- and post-menopausal conditions (32% and 30%, respectively). No differences were found concerning molecular subtype or histological grade (Table 3). In only 8 samples (14.55%), both RANK+ and RANKL+ tumor cells were identified, but in these overlapping cases H-scores were low. Based on the assessment of RANK and RANKL expression in stromal cells, 27 out of 56 evaluable samples (48,21%) and 18 out of 55 cases (32.7%) respectively exhibited an H-Score>0. Both the control and experimental groups were well balanced at baseline. There were no differences based on menopausal status; although in the premenopausal group stromal RANK expression was found in 57.7% compared to 40% in postmenopausal. Upon analysis by molecular subtype, it was noteworthy that luminal B-like tumors exhibited elevated RANK expression in the stroma, with rates of 75% compared to 34.5% in luminal A-like tumors and 30% in TNBC. This difference was statistically significant (p=0.012), representing the sole parameter where such distinction was observed (Table 3). Observing this pattern and recognizing that RANK and RANKL expressions behave more like continuous than categorical variables, we decided to perform an analysis between Ki67, histological grade, ER expression and TILs at the level of basal biopsies (to avoid deviations related to denosumab) and correlate them with the IHC expression of RANK and RANKL in these biopsies (Figure 6C-F). A positive correlation was identified between RANK expression in tumor cells and cell proliferation (Ki67) p=0.03 and histological grade p=0.015, while a negative correlation with ER expression p=0.006 and no association with % of TILs was observed (Figure 6C). The findings indicated that cases characterized by high histological grade, high Ki67 levels, and low estrogen receptor expression showed higher expression of RANK protein in tumor cells (Figure 6C). Despite tumors with the highest RANKL scores (H>25) showed low levels of ki67, RANKL expression in tumor cells did not associate with Ki67, nor with the other parameters (Figure 6D). Strikingly, RANK expression in the stroma was associated with high Ki67 expression p=0.001, while stromal RANKL did not associate with any parameter (Figure 6E-F). The percentage of TILs at baseline did not associate with RANK or RANKL expression. The notable correlation observed in between RANK expression in both tumor and stroma underlines its association with aggressive tumors. When restricting the analyses to luminal tumors neither RANK nor RANKL expression in tumor cells associated with any of the parameters analyzed (Supplementary data Figure S4A-B). Notably, increased stromal RANK expression remained associated with high Ki67 levels (p=0.001) and a positive correlation between RANKL in the stroma and TILs was observed (possibly as a marker of this population) (Supplementary data Figure S4 C-D). Therefore, we can infer that higher tumor proliferation correlates with higher RANK expression in the stroma in luminal tumors and the global analyses including TNBC. These results provide valuable information on the relationship between RANK, RANKL and other BC markers. As expected, no changes in the expression of RANK or RANKL in tumor cells or in the stroma were found between biopsy and surgery, neither in the control nor in the experimental arm (Supplementary data Figure S5A-D). Baseline RANK or RANKL expression did not predict denosumab-driven changes in TILs Univariate and multivariate analyses were performed to identify possible factors associated with the 10% increase in TILs. Only having a high Ki67 (>30) could be related to an elevation in TILS, despite no reaching significance in the multivariate analyses (OR 7.12 (1.18-43.1) (p=0.079) (Table 4). The only significant factor identified in the multivariate analysis that correlated with an increase in TILs was RANK expression in tumor cells at baseline (p < 0.001). No other factors were found to be significantly associated with the 10% increase in TILs (Table 4). Finally, when we analyzed the expression of RANK and RANKL as possible biomarkers of response to denosumab, we observed that the trends were similar to the overall population (Tables 5 and 6). There was no reduction in Ki67 or increase in cleaved caspase-3 after denosumab treatment when the analyses were performed only in tumors expressing RANK or RANKL protein in the tumor or stroma at baseline. Importantly, denosumab increased TILs regardless of tumor and stroma RANKL or RANK expression. This is, in tumor RANK positive samples, the experimental group showed an increase in TILs (p=0.013), similar to that observed in tumors that did not express RANK (tumor RANK-) (p=0.008). The same was observed when tumor RANKL expression was considered, the tumor RANKL positive group showed an increase in TILs (p=0.048), similar to the tumor RANKL negative group (p=0.002) (Table 6). The benefit of increased TILs after denosumab treatment was observed in tumors, irrespectively of the stromal expression of RANK or RANKL (Table 7). In conclusion, RANK and RANKL protein expression at baseline cannot be used as a biomarker capable of predicting the elevation of TILs caused by denosumab. 4. Discussion The D-BIOMARK trial was designed to investigate the biological effects of denosumab in patients with HER2-negative early breast cancer as a proof of concept for its potential antiproliferative, proapoptotic, and immunomodulatory effects on breast tumors and their microenvironment, beyond its bone-related effects. Our results are consistent with those of the D-BEYOND trial, which had a similar design but with a smaller number of patients, a single treatment arm, and only pre-menopausal women (29). The D-BIOMARK study overcomes these limitations by incorporating a control arm, encompassing both pre and postmenopausal patients, and including a larger number of TNBC patients. Preoperative denosumab did not reduce tumor cell proliferation or survival in early breast cancer. Subgroup analyses attending to menopausal status, surrogate molecular subtype and ER expression led to similar conclusions. However, we observed an effect on the immune response, with an increase in TILs exclusively in the experimental arm not only in pre-menopausal but also in post-menopausal patients, especially in the luminal B-like tumor subgroup. It is important to note that the RANK signaling has been identified as a crucial pathway in tumor initiation, inducing proliferation in normal epithelium and hyperplasia, but not in advanced lesions (ductal carcinoma in situ and adenocarcinomas) (2). These data are consistent with negative clinical results from D-BIOMARK and D-BEYOND regarding Ki67 and cleaved caspase-3. Once the tumor is established, the anti-proliferative and pro-apoptotic effect may be lost; however, there must be a mechanism, yet unclear, for those tumors with pharmacological or genetic inhibition of the pathway in preclinical studies to have fewer metastases (11,12). This therapeutic effect has also been reflected in clinical practice, with the large adjuvant trial ABCSG-18 demonstrating the benefit of adding denosumab in progression-free survival, bone metastasis-free survival, and overall survival, a trial designed exclusively in the luminal population (23). This benefit may be related to an anti-tumoral immune activation generated by denosumab, a hypothesis to be considered following our results. D-BIOMARK trial demonstrated the role of denosumab as an immune enhancer, evidenced by the increase of TILs found after treatment. Despite in the control group no significant increase in TILs was observed, we cannot discard that the inflammatory effect generated by the biopsy, or differences between the biopsy and surgery specimens may contribute to increase TILs, as both control and experimental arm showed the same trend of elevated TILs. However, it is important to note that also in the analyses by subgroups, the increase in TILs only reached significance in the denosumab-treated patients, including pre and postmenopausal, and luminal B-like tumors despite the lower number of samples. Although the increase in TILs is modest to have clinical relevance, it should be noted that patients only received 2 doses of denosumab with one week of interval, and the analysis by the different subgroups was consistent with the increase in TILs reported in the experimental arm. An important implication of these results is the potential use of denosumab as an enhancer of immune infiltration in neoadjuvant therapy for luminal B-like tumors, where immunotherapy with pembrolizumab (KEYNOTE-756 clinical trial) or nivolumab (CheckMate 7FL clinical trial) has shown to improve pCR but with rates close to 25%, much lower than in TNBC (39,40). Could denosumab improve these results? During neoadjuvant chemotherapy, initially elevated levels of TILs correlate positively with a higher rate of achieving a pCR in all breast cancer subtypes (41). A booster given by denosumab allowing new combinations of treatments in a tumor microenvironment that would otherwise be cold is of clinical interest. The GeparX study already combined denosumab with neoadjuvant chemotherapy without reporting additional benefits. Although an immediate impact on pCR may not be evident, the possibility of a long-term effect cannot be ruled out (25–28), and perhaps the best partner is the immunotherapy. There are already preclinical data showing synergy between immune checkpoint inhibitors (ICIs) and inhibitors of RANK signaling in solid tumors (29). The safety of the combination in clinical practice was reported in the CHARLI trial, a phase I/II study of the effect of denosumab with nivolumab (an anti-PD-1), with or without ipilimumab (anti-CTLA4), in patients with metastatic melanoma, which showed that the combination is safe and with at least interesting response rates (42). Pembrolizumab and denosumab have also been tested in clear cell renal cell carcinoma, a phase II trial (KeyPAD trial) with response rates close to 31% (43), and there are other ongoing trials with ICI in different tumors such as in lung cancer (Popcorn Trial) (44). Testing the combination in BC will be of interest, although we recognize that more data are required. Additionally, we have reported the expression of RANK and RANKL in tumor cells and stroma. A total of 34.54% of cases expressed RANK and 30% RANKL in the core biopsy, defined as an H-score > 0, slightly higher than reported in the literature, particularly in luminal tumors (18,19). In line with prior studies, RANK expression determined by IHC in tumor cells was associated with ER-negative tumors and high proliferative capacity (17–19). Our data confirm that RANK protein expression in tumor cells could serve as a biomarker for tumor aggressiveness: a correlation was observed between high Ki67, low ER expression, and high histological grade. Recent data from the GerpaX clinical trial showed that RANK expression in tumor cells was an independent predictive biomarker of response to neoadjuvant chemotherapy in luminal breast cancer, highlighting the opposite in TNBC and HER2-positive tumors (28). Apparently, and according to our data, luminal tumors might also have the RANK pathway involved in their pathophysiology, not only TNBC, where the biological effect of RANK has been reported (18,19,45). Strikingly our data reveals a novel association between RANK protein expression in the tumor microenvironment (stroma) with high levels of tumor proliferation (Ki67), which remains when studying exclusively luminal tumors (p=0.001). RANK expression in immune cells is predominantly found in myeloid cells such as macrophages and dendritic cells, while RANKL is predominantly found in TILs (20). Indeed, RANKL expression in the stroma of luminal tumors was associated with high percentage of TILs. Whether the highly proliferative tumors with RANK+ in the stroma have a greater immunosuppressive immune infiltrate is a hypothesis to be tested in the future. We were unable to establish a relationship between RANK or RANKL expression, neither in the stroma nor in the tumor cells, as a predictive marker for response to denosumab. However, RANK IHC in tumor cells was the only parameter in the multivariate analysis related to an increase of >10% in TILs between biopsy and surgery. This suggests that RANK expression in tumor cells may serve as a marker for aggressive tumors capable of recruiting higher levels of TILs. The difficulty in detecting RANK or RANKL as a response biomarker may be attributed to the variability and lack of standardization in the immunohistochemical technique for this staining, as well as the lack of a standardized cutoff point. In addition, the response, as evidenced by an increase in TILs after only two doses, was not strong enough to classify the change as a response, making it impossible to identify a biomarker. Regarding serum markers, the reduction in sRANKL after denosumab administration unequivocally confirms the inhibition of the RANK pathway in our study. However, the unexpected absence of changes in bone remodeling markers TRACP5b and CTX may be attributed to the short time interval between both samples and/or to the kinetics of the markers, a finding that contrasts with the results of the D-BEYOND trial (29). Nevertheless, the reduction in serum calcium levels in the experimental group serves as a surrogate marker of the impact on bone remodeling in the experimental arm. Additionally, there was an increase in OPG in the experimental group, related to the decrease in sRANKL, suggesting an incremental feedback loop or that denosumab binding to RANKL "displaces" OPG, increasing the detection of free OPG in circulation. Our analysis revealed a significant negative correlation between both markers. Surprisingly, post-menopausal women in our study displayed higher OPG levels, diverging from what is reported in the literature (46). The complexity of interpreting biomarker dynamics is underscored by factors such as bone mineral density, body mass index, and cardiovascular disease, which were not collected at enrollment (47,48). Additionally, sRANKL levels showed no association with age or menopausal status. These nuanced findings emphasize the need for comprehensive data collection to unravel the multifaceted influences on this pathway. As limitations of our study, we can highlight the small sample size limiting subgroup analyses and the tumor heterogeneity, especially affecting the cohort of TNBC, with a clear selection bias since most of these tumors receive neoadjuvant chemotherapy. This subgroup only includes 5 out of 10 typical aggressive TNBC, 2 cases with low proliferation index tumors and 3 cases of apocrine neoplasms. As strengths of our proposal, we have a control group that prevents erroneous conclusions; in addition, the inclusion of a higher number of samples allowed us not only to confirm the denosumab driven increase in TILs observed on tumors from premenopausal women, but also to extend these findings to postmenopausal BC and luminal B tumors. This study provides clinical validation of preclinical observations and previous studies, placing us in a closer reality of the actual effect of denosumab in early breast cancer (49). Consequently, our study serves as a crucial tool for understanding the behavior of this pathway within breast tumor cells, tumor microenvironment, and serum markers and opens new hypotheses for the implications of denosumab as a therapeutic target in BC, beyond its bone-related effects. This knowledge is essential for developing new drugs and for the more efficient utilization of denosumab. 5. Conclusions Two doses of denosumab before surgery did not reduce tumor proliferation or increase tumor apoptosis. However, this short course of denosumab increased TILs in early BC, particularly in luminal B-like tumors, and in pre-and postmenopausal BC. These findings suggest that denosumab may enhance the body's immune response against breast cancer, paving the way for further exploration and treatment refinement. Moreover, this trial underscores the complex nature of the RANK pathway, highlighting the necessity for further investigation into its multifaceted aspects. Declarations Funding: It’s an Investigator Sponsored Study: Amgen has assisted in the funding. The findings expressed herein are solely those of the authors, and Amgen does not assume any responsibility therefor. The study was sponsored by Institut Català d'Oncologia. Additionally, grants were provided by the European Research Council (ERC Consolidator No. 682935 to E. Gonzalez), the Ministry of Science, Innovation, and Universities (Juan de la Cierva contract IJCI-2017-31564 to EM Trinidad), the Carlos III Health Institute (Rio Hortega contract CM19/00148 to A Vethencourt), and the Spanish Breast Cancer Research Group GEICAM (Balil-Pelegrì grant to A Vethencourt). Institutional Review Board Statement: The study was conducted in accordance with the Declaration of Helsinki, and approved by the Institutional Ethics Committee of the “Institut Català d’Oncologia de L’Hospitalet (ICO-L’Hospitalet)” (protocol code PR035/21 ) Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Acknowledgments: We extend our heartfelt gratitude to all team members involved in the project, encompassing the medical oncology, radiology, pathology, and surgery teams. Special recognition is extended to Ariadna Iserte (ICO) for her exceptional coordination, and to Maria Dolores Mulero and the histology team at Idibell for their invaluable assistance with staining procedures. We also thank Emilia Brizzi and Marta Matas for their help with image interpretation and statistical analysis. Additionally, our appreciation extends to Cristina Moreno, Valentin Navarro and all individuals who contributed in various capacities. 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Osteoprotegerin concentration and risk of cardiovascular outcomes in nine general population studies: Literature-based meta-analysis involving 26,442 participants. PLoS One [Internet]. 2017 Aug 1 [cited 2023 Aug 8];12(8). Available from: /pmc/articles/PMC5570489/ Ciscar M, Trinidad EM, Perez-Montoyo H, Alsaleem M, Jimenez-Santos MJ, Toss M, et al. RANK is an independent biomarker of poor prognosis in estrogen receptor-negative breast cancer and a therapeutic target in patient-derived xenografts. bioRxiv [Internet]. 2021 Dec 14 [cited 2022 Mar 25];2021.12.13.470911. Available from: https://www.biorxiv.org/content/10.1101/2021.12.13.470911v1 Tables Tables 1 to 6 are available in the Supplementary Files section. Table 7 is not available with this version. Additional Declarations No competing interests reported. Supplementary Files TABLES.pptx SupplementaryData.pptx Cite Share Download PDF Status: Published Journal Publication published 12 May, 2025 Read the published version in Breast Cancer Research → Version 1 posted Editorial decision: Revision requested 12 Jan, 2025 Reviews received at journal 12 Oct, 2024 Reviewers agreed at journal 30 Sep, 2024 Reviewers agreed at journal 27 Sep, 2024 Reviewers agreed at journal 11 Jul, 2024 Reviewers invited by journal 17 May, 2024 Editor assigned by journal 22 Apr, 2024 Submission checks completed at journal 22 Apr, 2024 First submitted to journal 17 Apr, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4283385","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":294120258,"identity":"bdfb03b0-dc2d-4897-a3f6-967d2056b8bf","order_by":0,"name":"Andrea Vethencourt","email":"data:image/png;base64,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","orcid":"","institution":"Institut Català d'Oncologia","correspondingAuthor":true,"prefix":"","firstName":"Andrea","middleName":"","lastName":"Vethencourt","suffix":""},{"id":294120259,"identity":"3ae974dd-8f0b-4f62-86d9-5a24d30a8cc2","order_by":1,"name":"Eva M. 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Navarra","correspondingAuthor":false,"prefix":"","firstName":"Idoia","middleName":"","lastName":"Morilla","suffix":""},{"id":294120302,"identity":"628594b0-a5c8-472d-9cbe-f4da80bd7be6","order_by":14,"name":"Isaac Cachinero","email":"","orcid":"","institution":"Researchmar","correspondingAuthor":false,"prefix":"","firstName":"Isaac","middleName":"","lastName":"Cachinero","suffix":""},{"id":294120304,"identity":"45a32d0d-de52-4e7b-9dfb-07efeccc80cd","order_by":15,"name":"Amparo García-Tejedor","email":"","orcid":"","institution":"Bellvitge University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Amparo","middleName":"","lastName":"García-Tejedor","suffix":""},{"id":294120306,"identity":"0fc74f6e-0d09-45fc-87ae-00289e0cf6c8","order_by":16,"name":"Miguel Gil-Gil","email":"","orcid":"","institution":"Institut Català d'Oncologia","correspondingAuthor":false,"prefix":"","firstName":"Miguel","middleName":"","lastName":"Gil-Gil","suffix":""},{"id":294120307,"identity":"d8075660-cf1b-40fe-ad19-8e2e93cd1ab6","order_by":17,"name":"Sonia Pernas","email":"","orcid":"","institution":"Institut Català d'Oncologia","correspondingAuthor":false,"prefix":"","firstName":"Sonia","middleName":"","lastName":"Pernas","suffix":""},{"id":294120308,"identity":"94fa6139-994e-4d84-996b-f41a0d47d3cc","order_by":18,"name":"Catalina Falo","email":"","orcid":"","institution":"Institut Català d'Oncologia","correspondingAuthor":false,"prefix":"","firstName":"Catalina","middleName":"","lastName":"Falo","suffix":""},{"id":294120309,"identity":"1bc45492-1c5a-456e-bd0e-16f914590222","order_by":19,"name":"Eva Gonzalez-Suarez","email":"","orcid":"","institution":"IDIBELL, Institut d’Investigacio Biomédica de Bellvitge","correspondingAuthor":false,"prefix":"","firstName":"Eva","middleName":"","lastName":"Gonzalez-Suarez","suffix":""}],"badges":[],"createdAt":"2024-04-17 17:23:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4283385/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4283385/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13058-025-01996-w","type":"published","date":"2025-05-12T15:57:56+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":55538512,"identity":"0e9e5434-8f6a-4040-b0d3-4ad9eaeefdbd","added_by":"auto","created_at":"2024-04-29 16:50:06","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":107208,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eD-BIOMARK trial design\u003c/strong\u003e. Diagram showing the steps to be followed by a patient from the time the Informed Consent Form (ICF) is signed until the end of the study.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4283385/v1/32fb833c32621893067915a3.png"},{"id":55536324,"identity":"59e21919-0114-488f-8aa6-a8adc28f8778","added_by":"auto","created_at":"2024-04-29 16:34:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":100205,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCONSORT Flow diagram\u003c/strong\u003e. The CONSORT flow chart illustrates the flow of participants throughout the D-BIOMARK study. A total of 60 patients were initially enrolled, with 58 patients evaluated in the final analysis, 37 patients in the experimental arm and 21 patients in the control arm. The analysis was performed according to protocol.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4283385/v1/21da7ceb0c3d212cd190e6c0.png"},{"id":55536323,"identity":"1b30bcbb-1736-467f-9e73-aafe9e272bb8","added_by":"auto","created_at":"2024-04-29 16:34:06","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":486680,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSerum biomarkers: free RANKL (sRANKL), Osteoprotegerin (OPG), Tartrate-resistant acid phosphatase 5b (TRACP-5b) and Calcium.\u003c/strong\u003e Levels of sRANKL, OPG, TRACP-5b, detected by ELISA, and Calcium in serum from patients collected at the time of biopsy (Serum A) and surgery (Serum B) in the control and experimental arms. p value T-test for comparison between experimental and treatment arm is shown in the upper left corner and p value T-test for paired samples is shown for each treatment group\u003cstrong\u003e. \u003c/strong\u003eN indicates the number of samples analyzed. Note that Denosumab was associated with reduction in the levels of free RANKL and serum calcium, OPG shows a tendency to increase, while the levels of Trap5b did not change.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4283385/v1/6128cb8c1e0b35a49045ca73.png"},{"id":55537386,"identity":"174cdef4-90dd-4d96-b9cc-b64139fc216d","added_by":"auto","created_at":"2024-04-29 16:42:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":357501,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImpact of Denosumab on Tumor Cell Proliferation (Ki67) and apoptosis (cleaved-caspase 3): \u003c/strong\u003ePercentage of Ki-67+ tumor cells (A) and H-score of cleaved caspase-3 in tumor cells (B) at the diagnostic biopsy (biopsy) and in the surgical specimen (surgery) in the control and experimental arm. p value T-test for comparison between experimental and treatment arm is shown in the upper left corner and p value T-test for paired samples is shown for each treatment group\u003cstrong\u003e. \u003c/strong\u003eN indicates the number of samples analyzed. Note than both the experimental and control groups exhibited an increase in the percentage of Ki67-positive cells for paired samples and Denosumab did not induce an increase in tumor cell apoptosis. Comparable mean levels of Ki67 and cleaved caspase-3 were observed between both groups.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4283385/v1/6d49ea53b7390d8267a9d2a7.png"},{"id":55537384,"identity":"9bdd8972-1bb5-40d7-9b72-1160cc700e42","added_by":"auto","created_at":"2024-04-29 16:42:06","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":537298,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImpact of Denosumab on stromal Tumor Infiltrating Lymphocytes (TILs). \u003c/strong\u003eA: Percentage of stromal TILs at the diagnostic biopsy (biopsy) and in the surgical specimen (surgery) in the control and experimental arm. p value T-test for comparison between experimental and treatment arm is shown in the upper left corner and p value T-test for paired samples is shown for each treatment group\u003cstrong\u003e. \u003c/strong\u003eN indicates the number of samples analyzed. Note than only the experimental group exhibited an increase in the percentage of TILs B. Representative image of H\u0026amp;E staining depicting changes in TILs between biopsy and surgery in patients from the experimental arm in an invasive carcinoma (NOS) luminal B-like.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4283385/v1/d2da9cccdb760a1f8712892a.png"},{"id":55536328,"identity":"5ecec266-f19f-4bc5-b295-e0679f242745","added_by":"auto","created_at":"2024-04-29 16:34:07","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":550481,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCorrelation analysis between RANK/RANKL expression in tumor or stroma and clinicopathological parameters. A-B \u003c/strong\u003eRepresentative image of RANK (A) and RANKL (B) staining\u003cstrong\u003e. \u003c/strong\u003eC-F. Correlation analysis between RANK/RANKL expression in tumor or stroma with ki67, histological grade, ER and TILs using Pearson's correlation coefficient. RANK expression in tumor cells positively correlates with Ki67, histological grade, and negatively correlates with estrogen receptor expression. Additionally, RANK expression in stroma is associated with high Ki67 levels. No significant correlation was found with RANKL expression.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4283385/v1/1392c11f1e7c2dcccf6cd657.png"},{"id":83068091,"identity":"03d01357-fecb-4427-88ae-f4fb5f983709","added_by":"auto","created_at":"2025-05-19 16:10:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3067712,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4283385/v1/8b97bb23-8bf6-4c05-9646-84ffcfe677d5.pdf"},{"id":55536325,"identity":"3f9fc96a-862f-4c4e-84e2-b3a16acd3d9a","added_by":"auto","created_at":"2024-04-29 16:34:06","extension":"pptx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":102883,"visible":true,"origin":"","legend":"","description":"","filename":"TABLES.pptx","url":"https://assets-eu.researchsquare.com/files/rs-4283385/v1/c89bfba595af2180f6873371.pptx"},{"id":55536329,"identity":"fdea2a56-9a6c-4fa0-8e52-13d9fc48383c","added_by":"auto","created_at":"2024-04-29 16:34:07","extension":"pptx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":990379,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryData.pptx","url":"https://assets-eu.researchsquare.com/files/rs-4283385/v1/b712f6cbe1e5df710e2538d4.pptx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Denosumab as an immune modulator in HER2-negative early breast cancer: results of the window-of-opportunity D-BIOMARK clinical trial.","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe receptor activator of nuclear factor \u0026kappa;B, known as RANK, member of tumor necrosis factor receptor (TNFR) superfamily, and its ligand (RANKL) \u0026nbsp;have emerged as potential therapeutic targets in breast cancer (BC) and other solid tumors (1\u0026ndash;3).\u003c/p\u003e\n\u003cp\u003eBinding of RANKL to RANK leads to activation of signaling pathways related to tumor proliferation, survival, and inflammation, such as the canonical and non-canonical NF\u0026kappa;B, MAPK and PI3K-AKT. Osteoprotegerin (OPG), a natural negative regulator of the RANK pathway acts as a decoy receptor and prevents the binding of RANKL to RANK. The RANK/RANKL/OPG axis has been widely studied as a regulator of bone resorption, leading to the development of denosumab, a highly specific immunoglobulin type IgG2 monoclonal antibody, which binds with high affinity to human RANKL and neutralizes its activity (4). Denosumab is approved for the prevention of skeletal events in patients with bone metastases and for the treatment of unresectable giant cell tumors of bone and osteoporosis (3,5,6).\u003c/p\u003e\n\u003cp\u003ePreclinical data show that RANK signaling regulates mammary gland development and mammary cell fate (7\u0026ndash;10). RANKL is the main mediator of the proliferative and pro-tumorigenic role of progesterone in the mammary gland. Pharmacological or genetic inhibition of the pathway prevents or attenuates mammary tumor appearance, reduces cell proliferation in preneoplastic lesions and tumor cell survival in mouse adenocarcinomas (2,11). Blockade of the RANK pathway also reduces the incidence of lung metastases and enhances the differentiation of tumor cells in mouse models (2,11\u0026ndash;14).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn human mammary adenocarcinomas, RANK protein expression, detected by immunohistochemistry (IHC), is found in 15-20% of estrogen receptor (ER)-positive tumors and in 40% of ER-negative breast adenocarcinomas. RANK protein expression in tumor cells associates with an aggressive tumor phenotype, including hormone receptor-negative tumors, high histological grade and high proliferative index (15\u0026ndash;19). Recent findings underscore that RANK protein expression in tumor cells serves as an independent maker of adverse prognosis in post-menopausal patients and ER-negative BC (19).\u003c/p\u003e\n\u003cp\u003eRANK signaling plays a crucial role in regulating the delicate balance between tolerance and immunity (20). RANK is predominantly expressed by myeloid cells such as dendritic cells and macrophages while RANKL expression has been observed on T cells in both the tumor microenvironment (TME) and locoregional lymph nodes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDespite strong preclinical evidence, the therapeutic benefit of denosumab in BC patients beyond its bone-related effects is unclear. In the adjuvant setting, the phase III ABCSG-18 clinical trials clinical trial demonstrated that administering denosumab at 60 mg every 6 months for 5 years, alongside an aromatase inhibitor, not only delayed bone fractures and improved mineral bone density but also increased 8-year disease-free survival (DFS) from 77.5% to 80.6%, with a hazard ratio (HR) of 0.82 (95% confidence interval CI=0.69-1.98), p=0.02. In 2022, further analysis confirmed these benefits, including progression-free survival, bone metastasis-free survival, and overall survival, leading to a recommendation for routine use of denosumab as adjuvant therapy in post-menopausal women with hormone receptor-positive BC (21\u0026ndash;23). In contrast, in the D-CARE clinical trial, with adjuvant denosumab at a dose of 120 mg every 4 weeks during 6 months and then every 12 weeks up to five years, did not show changes in bone metastasis-free survival (HR 0.97 (95% IC 0.82-1.14), p=0.70) and reported a similar 5-year DFS between the two groups (HR: 1.04 (95% IC 0.91-1.19), p=0.57) (24). Both studies involved different populations and yielded discordant results. In the neoadjuvant context in the GeparX study, the addition of denosumab to neoadjuvant treatment did not increase the pathological complete response (pCR) rate in early BC (25\u0026ndash;28).\u003c/p\u003e\n\u003cp\u003eThe D-BEYOND, a prospective single-arm window-of-opportunity trial, evaluated the biological effect of denosumab in pre-menopausal women diagnosed with early BC. It included 27 patients, only one was triple negative breast cancer (TNBC). All patients received two subcutaneous injections of denosumab (120 mg/dose) separated by one week prior to breast surgery. The baseline biopsies were compared with the surgical sample. The study did not meet its primary aims: a reduction in cell proliferation nor an increase in cell apoptosis was observed. However, a brief course of denosumab induced an increase in the inflammatory infiltrate measured by tumor-infiltrating lymphocytes (TILs), especially CD8+ T lymphocytes (29).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHere, we present results from the D-BIOMARK trial (NCT03691311), a window-of-opportunity study designed to assess the biological activity of single-agent denosumab in patients with primary operable HER2-negative BC.\u0026nbsp;\u003c/p\u003e"},{"header":"2. Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003e2.1 Trial design and patients\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eD-BIOMARK (NCT03691311) is a prospective, single institution, randomized window-of-opportunity clinical trial evaluating the biological effects of single-agent denosumab in treatment-naive patients with early HER2-negative BC who were candidates for tumor excision as the first therapeutic approach. Exclusion criteria included osteonecrosis of the jaw or risk of developing it, other active malignancies, hypocalcemia, or known hypersensitivity to denosumab. Randomization was stratified by menopausal status and ER-status (ER+ \u003cem\u003evs\u003c/em\u003e triple negative). Post-menopausal was defined clinically as more than 1 year with amenorrhea, or \u0026ge; 60 years old (30). Early-stage breast cancer is defined as stage I, stage IIA, stage IIB, and stage IIIA breast cancers, which denote cancer that has not spread beyond the breast or the axillary lymph nodes (31).\u003c/p\u003e\n\u003cp\u003eWritten informed consent was obtained from all patients prior to any procedure within the study. Patients were randomized 2:1 to the experimental or control arm; the experimental group received two subcutaneous doses of denosumab (120 mg each) administered 7 days apart, while the control group received no treatment. Screening visits were conducted for all participants. In both arms, a biopsy was performed at the time of diagnosis (referred to as \u0026quot;biopsy\u0026quot;), with serum collected also at baseline (referred to as \u0026quot;serum A\u0026quot;). Furthermore, a secondary biopsy of the surgical specimen was performed two to four weeks after enrollment, at the time of the surgical excision of the breast tumor (referred to as \u0026quot;surgery\u0026quot;), accompanied by a second blood sample collection (referred to as \u0026quot;serum B\u0026quot;). Additional visits and blood analyses were conducted in the experimental arm before each treatment session. Both groups underwent two follow-up visits for safety assessment at one month and six months post-surgery (Figure 1). Patients in the experimental group received daily calcium supplements (\u0026ge; 500 mg elemental calcium) and vitamin D (\u0026ge; 400 IU) for one month following the first dose to prevent hypocalcemia.\u003c/p\u003e\n\u003cp\u003eThe study protocol was approved by the institutional ethics committee (Research Ethics Committee of the Hospital Universitari De Bellvitge) protocol code PR035/21 and conducted in accordance with the ethical standards outlined in the 1964 Declaration of Helsinki.\u003c/p\u003e\n\u003cp\u003eClinical data and biological characteristics of the tumors were prospectively obtained from medical records and pathology reports, respectively. Adverse events were recorded starting from the day of obtaining signed informed consent and continued until six months following surgery. The safety data were assessed in accordance with the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE v5.0).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Tumor assessment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe evaluation of conventional BC markers, including ER, progesterone receptor (PR), HER2, and Ki67, was performed in the Pathology Department of the Hospital Universitari de Bellvitge. The status of ER and PR was defined according to the guidelines of the American Society of Clinical Oncology and the College of American Pathologists (ASCO-CAP 2010) (32). The histological grade was evaluated following Nottingham classification (33). The surrogate subtypes of BC were defined according to St Gallen 2015 consensus meetings, using IHC substitutes as follows: luminal A-like: ER and/or PR(+), HER2(-), Ki67 \u0026lt; 20%; luminal B-like: ER and/or PR(+), HER2(-), Ki67 \u0026ge; 20; TNBC: ER(-), PR(-), and HER2(-), regardless of the Ki67 score (34).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe objectives of this trial were tested by comparing the biopsy at diagnosis (biopsy) with the surgical specimen (surgery). The evaluation of tumor cellularity was assessed in hematoxylin-eosin (H\u0026amp;E) staining tissue sections. For patients with multiple samples, the sample with the highest tumor content was chosen. The percentage of Ki67, cleaved caspase-3 and TILs were independently evaluated by at least two pathologists specialized in BC, blinded to clinical and experimental data. For Ki67, the antibody used was MIB-1, Agilent Dako, the fixation conditions, processing, and results evaluation were performed according to international recommendations (35,36). TILs were evaluated on H\u0026amp;E-stained slides using standardized methodology (37). The anti‐cleaved caspase‐3 was assessed using Asp175, Cell Signaling; 1:200; the quantification of cleaved caspase-3, as an experimental parameter, has no international quantification guidelines; by recommendation of expert pathologists, it was performed using QuPath\u0026reg; bio-image analysis software and a H-score = (% of cells with weak intensity \u0026times; 1) + (% of cells with moderate staining \u0026times; 2) + (% of cells with strong staining \u0026times; 3). The maximum possible H-score is 300, corresponding to 100% of cells with strong (3+) intensity.\u003c/p\u003e\n\u003cp\u003eTo evaluate RANK and RANKL, paraffined tissue sections (4 \u0026mu;m) were used. For each patient, representative unstained slides of the tumor were shipped to NeoGenomics Laboratories (California, USA) for IHC staining of RANK (N1H8, Amgen), RL (M366, Amgen), blinded to clinical information. The percentage of stained cells and their intensity (0, negative; 1+, weak; 2+, moderate; and 3+, strong) in the tumor cells were reported by NeoGenomics, and the H-score was calculated using the previous formula. Due to the complexity of RANK and RANKL IHC interpretation and its reading, a double evaluation of H-scores in tumor cells was performed (NeoGenomics and at the laboratory of Dr. Gonzalez-Suarez). In addition, RANK and RANKL H-scores in stroma were evaluated only at the laboratory of Dr. Gonzalez-Suarez (2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 Serum analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSerum concentrations of human free RANKL (sRANKL), tartrate-resistant acid phosphatase 5b (TRACP5b), carboxy-terminal collagen crosslinks (CTX), and OPG were quantified utilizing an enzyme-linked immunosorbent assay (ELISA), in accordance with the manufacturer\u0026rsquo;s guidelines. Progesterone, estradiol, follicle-stimulating hormone (FSH or Follitropin), calcium, albumin, and blood count values were extracted from laboratory reports processed at the Hospital Universitari de Bellvitge laboratory, where the patient\u0026apos;s routine tests were performed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 Statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe total patient sample size was defined as 60: 40 in the treatment arm and 20 in the control arm. \u0026nbsp; Since there were no prior data on the distribution, mean and standard deviation of the primary endpoint available at the time of the protocol design, a conventional statistical design for sample size calculation was not feasible; therefore, the trial was designed according to Larry V. Rubinstein\u0026apos;s suggestions for phase 0 trials\u0026nbsp;(38). \u0026nbsp;For biological purposes, a minimum of 10 TNBC tumors and a minimum of 24 pre-menopausal patients were included. \u0026nbsp;Assignment to each arm was done by stratified block randomization.\u003c/p\u003e\n\u003cp\u003eAll analyses were performed using R version 4.1.3 (available at www.r-project.org), GraphPad Prism version 5 and IBM SPSS Statistics version 25 (IBM Corp, Armonk, NY, USA). At baseline (biopsy/serum A) \u003cem\u003evs.\u003c/em\u003e at surgery (surgery/serum B) values were compared using a paired t-test or McNemar test for numeric and binary variables, respectively. Independent samples t-test was used to compare differences between groups, while chi-squared or Fisher exact test, when appropriate, was used for binary variables.\u003c/p\u003e\n\u003cp\u003eTo compare baseline variables and possible predictive factors for response to denosumab, the Mann\u0026ndash;Whitney U and Fisher\u0026rsquo;s exact tests were used for continuous and categorical variables, respectively. All correlations were measured using Spearman\u0026rsquo;s non-parametric rho coefficient. A Logistic regression analysis was performed to define the odds ratio of developing a response variable. All reported \u003cem\u003ep\u003c/em\u003e-values were two-tailed. Statistical significance was set at 0.05.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1\u003c/strong\u003e \u003cstrong\u003ePatient Population\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 60 patients were enrolled in the study between August 2018 and May 2021. Two patients were excluded. \u0026nbsp;One of them received neoadjuvant letrozole during the SarsCov2 pandemic period due to a delay in the scheduled date of surgery, and other patient only received one dose of denosumab due to withdrawal of consent after the first infusion. One patient who underwent all the procedures but did not receive denosumab due to an administrative error was transferred to the control group. Thus, a total of 37 patients were analyzed in the experimental group and 21 patients in the control group, all 58 patients completed the follow-up period (Figure 2). The analysis was done by protocol.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe clinicopathological baseline characteristics of the evaluable patients are shown in Table 1. The mean age of the study population was 56.4 years (range, 37-80 years), with a mean age of 57 years in the experimental arm and 55.4 years in the control arm. The mean time between the first administration of denosumab and surgery was 21 days. Of the total participants, 27 were pre-menopausal women, with 17 (45.9%) in the experimental arm and 10 (47.6%) in the control group, meeting the requirement of including at least 24 pre-menopausal women. The distribution of post-menopausal patients was: 20 (54.1%) in the experimental arm and 11 (52.4%) in the control group. Most patients were classified as IA clinical stage, with 26 cases (70.3%) in theT experimental arm and 17 cases (81%) in the control arm. The median tumor size was 18 mm (range 8-45 mm).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn terms of tumor characteristics, the majority of patients had invasive breast cancer without specific features, classified as no special type (NST) or not otherwise specified (NOS) (ductal), 62.07% overall; with a slightly higher percentage in the control arm (71.4% vs. 56.8% in the experimental group), with a lower number of patients with invasive lobular carcinoma in the control arm (14.3 vs. 32.4% in the experimental arm), although these differences were not significant. Most tumors had a histological grade 2 (58.62%), with 7 tumors (12.06%) classified as grade 3; notably, there was a higher percentage of cases with histological grade 1 tumors (35.1%) in the experimental group compared to the control group (19%). The data are consistent with a higher number of cases with low Ki67 (\u0026lt;15) in the experimental group (43.2%) compared to the control group (9.5%). Ki67 was the only parameter that showed significance (p=0.001) when comparing both groups; however, when analyzing the tumors by surrogate molecular subtype, this difference was not significant, although numerically, there were still more cases of luminal A-like tumors in the experimental arm (51.4% vs. 38.1%). The groups were well balanced as no statistically significant differences between experimental and control arms were observed, except for the percentage of patients with tumors with low Ki67 (\u0026lt;15%) (Table 1). Of the total of 58 evaluable cases, 48 were luminal tumors (27 luminal A-like and 21 luminal B-like), while 10 were TNBC. Initially, a higher percentage of TNBC cases was expected. However, most TNBC tumors were selected for neoadjuvant treatment and were therefore excluded from our study. Upon reviewing these 10 TNBC cases, only 5 exhibited typical aggressive characteristics (histological grade 2/3, Ki67 \u0026gt;30%), with 3 of these cases in the control group and 2 in the experimental arm. The remaining cases were low aggressive TNBC, including 3 cases of invasive carcinomas with apocrine differentiation, 2 in the experimental arm and 1 in the control arm: apocrine carcinomas are known to exhibit a more indolent behavior compared to typical TNBCs. Additionally, the experimental group had 1 case of lobular carcinoma and 1 case of carcinoma not otherwise specified (NOS) ductal, with Ki67 of 10% and 5%, respectively. Given these factors, caution should be exercised in drawing conclusions about the triple negative subgroup.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Safety Data\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFive patients experienced localized hematomas in the breast following study biopsies, which were classified as grade 1 and did not require drainage or special measures; 2 of these cases were in the control group and 3 were in the experimental group. In the experimental arm, the most common adverse events were grade 1 or 2 bone pain occurring 24 hours after infusion in 10 of 37 patients (27.03%), grade 1 asthenia in 4 patients (10.81%), grade 1 pain at the denosumab infusion site in 3 patients (8.10%), grade 1 chills in 2 patients (5.41%), and grade 2 dental infection in 1 patient (2.70%). There were no reported cases of hypocalcemia. No grade 3 toxicities were reported (see Table S1 in the Supplementary Appendix).\u003c/p\u003e\n\u003cp\u003eOutside the study follow-up period, we observed a long-term event of osteonecrosis of the jaw in a heavy-smoker patient who received denosumab on March 10\u003csup\u003eth\u003c/sup\u003e and 17\u003csup\u003eth\u003c/sup\u003e, 2021. This patient was initially reported with a dental infection. Symptoms began one month after the last dose of denosumab (reported on April 19, 2021), with discomfort and pain in the jaw, the case was referred to the Maxillofacial Surgery Department. Computed tomography initially did not reveal signs of osteonecrosis, and the case was initially diagnosed as a tooth infection, which improved with oral antibiotic treatment. However, repeated episodes of dental infection in the same location needed specific follow-up. After 11 months, the patient underwent jaw surgery in February 2022, and the pathological report confirmed osteonecrosis of the right quadrant 44-47. This event was considered possibly related to denosumab, although other triggering risk factors such as chronic infection due to long-term smoking should be considered. The osteonecrosis of the jaw was classified as grade 3. At the time of this report, no grade 4 or 5 toxicity has been reported.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 Serum Analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDenosumab was associated with systemic inhibition of RANKL but not with changes in bone remodeling markers\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe blockade of the RANK-RANKL pathway was confirmed by the drop in serum of free RANKL (sRANKL), measured by ELISA, in the experimental group as RANKL became bound to denosumab (mean serum A 0,096 pg./L vs serum B vs. 0,000 pg./L p\u0026lt;0.001), while no changes were found in the control group (mean serum A 0,100 pg./L vs. serum B 0,116 pg./L; p=0.270) comparing serum A vs serum B.\u0026nbsp;OPG levels tended to increase in the experimental group (p=0.071), consistent with the reduction of free RANKL.\u0026nbsp;However, the serum levels of the bone resorption markers\u003cstrong\u003e\u0026nbsp;t\u003c/strong\u003eartrate-resistant acid phosphatase 5b (TRACP5b) (n=37) and carboxy-terminal collagen crosslinks (CTX) (n=38) did not change in any group (Figure 3 and Supplementary data Figure S1). It is unclear whether the lack of changes is due to technical limitations (limited detection rate) or due to the kinetics of bone resorption. Although no significant alterations were observed in bone resorption markers, a small decrease in serum calcium -not clinically relevant- was reported in the experimental arm (Figure 3), despite the prescribed calcium and vitamin D supplementation. This finding reinforces the efficacy of denosumab in bone remodeling.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA correlation analysis was conducted between the different serum markers studied and menopausal status to better understand the biology of the pathway. Follicle-stimulating hormone (FSH) levels were higher in post-menopausal patients, consistent with menopausal physiology (p\u0026lt;0.0001). Levels of free sRANKL did not differ based on menopausal status, while higher levels of OPG were detected in post-menopausal women (p=0.010) at baseline (Supplementary data Figure S2A). Comparable values of the bone markers TRACP5b, and CTX were found at the time of diagnosis (baseline) between pre- and post-menopausal women, although slightly higher levels were found in the post-menopausal group. In pre-menopausal patients, no associations were found between levels of progesterone and sRANKL in serum (p=0.401). Correlation between OPG and TRACP5b values was not significant, unless one sample with high OPG and low TRACP5b was excluded. Finally, a negative correlation between OPG and sRANKL was demonstrated (p=0.0026), consistent with the known interaction between these factors (Supplementary data Figure S2B).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eTumor assessment\u0026acute;s results\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDenosumab was not associated with a reduction in tumor cell proliferation or an increase in apoptosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe primary endpoints of the clinical trial were a decrease in tumor cell Ki67 and an increase in apoptosis between biopsy and surgery. Denosumab did not reduce tumor cell proliferation or survival between paired biopsy and surgery samples (Figure 4A-B). The percentage of tumor cells expressing Ki67 increased in both groups (control p=0.035 and experimental p=0.012), which may be attributed to a higher quantification of fields within the surgical specimen (more fields) compared to the core biopsy. The mean Ki67 in the control arm increased from 24.52% at biopsy to 29.19% at surgery. Similarly, in the experimental arm, it increased from 20.86% to 24.81%. This nearly 5-percentage-point increase when comparing surgical samples with baseline biopsies in both groups suggests that these changes were not influenced by denosumab treatment. Indeed, a comparison between the experimental and control groups revealed identical behavior (p=0.928) (Figure 4A).\u003c/p\u003e\n\u003cp\u003eDenosumab did not induce an increase in tumor cell apoptosis, as demonstrated by the assessment of the H-score of cleaved caspase-3 between biopsy and surgery. The comparison between the experimental and control groups revealed notable inter-patient variability. Although a statistically significant difference in apoptosis was observed inter-group (p=0.042), the change in cleaved caspase-3 H-score quantification was less than 1 in both groups. Despite the contrasting trends, the alteration in apoptosis is considered clinically insignificant due to very low H-scores in all cases. The evaluating intra-patient (Paired T-test) showed no changes (control group p=0.060 and experimental p=0.238). Additionally, 3 patients in the experimental group exhibited higher levels of cleaved caspase-3 at baseline (Figure 4B) that may suggest potential deterioration or non-specific staining in some areas.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA subgroup analysis was conducted to elucidate if any specific patient group could benefit from denosumab treatment. Patients were divided according to surrogate molecular subtype (Supplementary data Table S2). Patients with luminal A-like (n=27) and luminal B-like (n=21) tumors showed similar trends to the overall population: there was no reduction in Ki67 or increase in Cleaved Caspase-3 in the experimental group and no changes were observed in OPG or TRACP5b. In the TNBC group (n=10), there was an increase in Ki67 in the experimental arm (p=0.025), which was not evident in the control group (p=0.517), but no difference was found in the inter-group comparison (p=0.197). Moreover, it is important to note the small number of TNBC cases, with 6 in the experimental group and 4 in the control group, as well as an imbalance between aggressive tumors, as previously explained (Supplementary data Table S2).\u003c/p\u003e\n\u003cp\u003eA subgroup analysis was also conducted based on menopausal status, excluding cases of triple-negative tumors to avoid biasing the information, as 9 out of 10 TNBC tumors were post-menopausal. Both pre-menopausal (n=26) and post-menopausal (n=22) tumors showed similar trends, with no reduction in proliferation or cell survival, and no other notable findings (Supplementary data Table S3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDenosumab increased tumor infiltrating lymphocytes in early breast cancer, particularly in luminal B-Like tumors and regardless of menopausal status\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, we interrogated the effect of denosumab on tumor immune infiltration and observed an increase in TILs in the surgery sample compared to the initial biopsy in the experimental arm (p=0.001), but not in the control arm (p=0.06). However, both groups showed a similar trend (p=0.789) (Figure 5A-B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePerforming a similar analysis based on surrogate molecular subtype, we found that while no changes in TILs were noted in Luminal A tumors (experimental (p=0.144), control (p=0.958)), there was a denosumab-induced elevation in TILs in luminal B-like tumors (p=0.012), with no significant changes in the control group (p=0.070). TILs did not change in TNBC (experimental (p=0.079), control (p=0.237)), although an increased number of representative TNBC tumors is required to reach conclusions in this subtype. Attending to menopausal stage in luminal tumors, we found that both pre-menopausal and postmenopausal patients experienced an increase in TILs in the experimental arm (premenopausal: p=0.048, postmenopausal: p=0.041), but not in the control arm (premenopausal: p=0.639, postmenopausal: p=0.062). In all comparisons the experimental and control arms showed similar trends (Table 2). Furthermore, applying a threshold of a 10% or greater increase in TILs between biopsy and surgery samples, we observed that 9 out of 37 (24.3%) patients in the experimental arm and 5 out of 21 (23.8%) patients in the control arm showed a clinically significant increase in TILs.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTumor and stroma RANK expression was associated with highly proliferative tumors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe quantification conducted externally by NeoGenomics and in-house by the laboratory of Dr. Gonzalez-Suarez showed perfect correlation in the analysis of tumor RANK and RANKL protein expression (Supplementary data Figure S3). Given the correlation and the fact that the quantification of H-score for RANK and RANKL in the stroma was only conducted in the laboratory of Dr. Gonzalez-Suarez, all subsequent analyses were carried out using in-house quantification.\u003c/p\u003e\n\u003cp\u003eA total of 55 cases were assessable for tumor RANK and RANKL expression at baseline (Table 3, Figure 6A-B) and 19 tumors (34.54%) exhibited positive baseline expression of RANK, defined as an H-score \u0026gt; 0 (tumor RANK+). Of these positive cases, 14 tumors were randomized to the experimental arm (38.9%), and 5 were assigned to the control group (p=0.526, well balanced). The frequency of RANK+ tumors was comparable between tumors from premenopausal (36%) and postmenopausal patients (33%). Additionally, we compared tumor RANK expression across different molecular subtypes, 28% of luminal A-like, 35% of luminal \u0026ndash;B-like and 50% of triple negative tumors exhibited RANK+ tumor cells, a higher frequency than that previously reported, particularly in luminal tumors (18,19). A total of 42.4% of grade 2 and 42.9% of grade 3 tumors were positive for tumor RANK, compared to only 13.3% of grade 1 tumors.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRegarding RANKL expression, 17 out of 55 tumors (30.10%) exhibited RANKL expression in tumor cells, with 12 assigned to the experimental group and 5 to the control group (p=0.819, well balanced). The frequency of tumor RANKL expression was similar between pre- and post-menopausal conditions (32% and 30%, respectively). No differences were found concerning molecular subtype or histological grade (Table 3). In only 8 samples (14.55%), both RANK+ and RANKL+ tumor cells were identified, but in these overlapping cases H-scores were low.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBased on the assessment of RANK and RANKL expression in stromal cells, 27 out of 56 evaluable samples (48,21%) and 18 out of 55 cases (32.7%) respectively exhibited an H-Score\u0026gt;0. Both the control and experimental groups were well balanced at baseline. There were no differences based on menopausal status; although in the premenopausal group stromal RANK expression was found in 57.7% compared to 40% in postmenopausal. Upon analysis by molecular subtype, it was noteworthy that luminal B-like tumors exhibited elevated RANK expression in the stroma, with rates of 75% compared to 34.5% in luminal A-like tumors and 30% in TNBC. This difference was statistically significant (p=0.012), representing the sole parameter where such distinction was observed (Table 3).\u003c/p\u003e\n\u003cp\u003eObserving this pattern and recognizing that RANK and RANKL expressions behave more like continuous than categorical variables, we decided to perform an analysis between Ki67, histological grade, ER expression and TILs at the level of basal biopsies (to avoid deviations related to denosumab) and correlate them with the IHC expression of RANK and RANKL in these biopsies (Figure 6C-F). A positive correlation was identified between RANK expression in tumor cells and cell proliferation (Ki67) p=0.03 and histological grade p=0.015, while a negative correlation with ER expression p=0.006 and no association with % of TILs was observed (Figure 6C). The findings indicated that cases characterized by high histological grade, high Ki67 levels, and low estrogen receptor expression showed higher expression of RANK protein in tumor cells (Figure 6C). Despite tumors with the highest RANKL scores (H\u0026gt;25) showed low levels of ki67, RANKL expression in tumor cells did not associate with Ki67, nor with the other parameters (Figure 6D). Strikingly, RANK expression in the stroma was associated with high Ki67 expression p=0.001, while stromal RANKL did not associate with any parameter (Figure 6E-F). The percentage of TILs at baseline did not associate with RANK or RANKL expression. The notable correlation observed in between RANK expression in both tumor and stroma underlines its association with aggressive tumors.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWhen restricting the analyses to luminal tumors neither RANK nor RANKL expression in tumor cells associated with any of the parameters analyzed (Supplementary data Figure S4A-B). Notably, increased stromal RANK expression remained associated with high Ki67 levels (p=0.001) and a positive correlation between RANKL in the stroma and TILs was observed (possibly as a marker of this population) (Supplementary data Figure S4 C-D). Therefore, we can infer that higher tumor proliferation correlates with higher RANK expression in the stroma in luminal tumors and the global analyses including TNBC. These results provide valuable information on the relationship between RANK, RANKL and other BC markers.\u003c/p\u003e\n\u003cp\u003eAs expected, no changes in the expression of RANK or RANKL in tumor cells or in the stroma were found between biopsy and surgery, neither in the control nor in the experimental arm (Supplementary data Figure S5A-D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBaseline RANK or RANKL expression did not predict denosumab-driven changes in TILs\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUnivariate and multivariate analyses were performed to identify possible factors associated with the 10% increase in TILs. Only having a high Ki67 (\u0026gt;30) could be related to an elevation in TILS, despite no reaching significance in the multivariate analyses (OR 7.12 (1.18-43.1) (p=0.079) (Table 4). The only significant factor identified in the multivariate analysis that correlated with an increase in TILs was RANK expression in tumor cells at baseline (p \u0026lt; 0.001). No other factors were found to be significantly associated with the 10% increase in TILs (Table 4).\u003c/p\u003e\n\u003cp\u003eFinally, when we analyzed the expression of RANK and RANKL as possible biomarkers of response to denosumab, we observed that the trends were similar to the overall population (Tables 5 and 6). There was no reduction in Ki67 or increase in cleaved caspase-3 after denosumab treatment when the analyses were performed only in tumors expressing RANK or RANKL protein in the tumor or stroma at baseline. Importantly, denosumab increased TILs regardless of tumor and stroma RANKL or RANK expression. This is, in tumor RANK positive samples, the experimental group showed an increase in TILs (p=0.013), similar to that observed in tumors that did not express RANK (tumor RANK-) (p=0.008). The same was observed when tumor RANKL expression was considered, the tumor RANKL positive group showed an increase in TILs (p=0.048), similar to the tumor RANKL negative group (p=0.002) (Table 6). The benefit of increased TILs after denosumab treatment was observed in tumors, irrespectively of the stromal expression of RANK or RANKL (Table 7). In conclusion, RANK and RANKL protein expression at baseline cannot be used as a biomarker capable of predicting the elevation of TILs caused by denosumab.\u0026nbsp;\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe D-BIOMARK trial was designed to investigate the biological effects of denosumab in patients with HER2-negative early breast cancer as a proof of concept for its potential antiproliferative, proapoptotic, and immunomodulatory effects on breast tumors and their microenvironment, beyond its bone-related effects. Our results are consistent with those of the D-BEYOND trial, which had a similar design but with a smaller number of patients, a single treatment arm, and only pre-menopausal women (29). The D-BIOMARK study overcomes these limitations by incorporating a control arm, encompassing both pre and postmenopausal patients, and including a larger number of TNBC patients. Preoperative denosumab did not reduce tumor cell proliferation or survival in early breast cancer. Subgroup analyses attending to menopausal status, surrogate molecular subtype and ER expression led to similar conclusions. However, we observed an effect on the immune response, with an increase in TILs exclusively in the experimental arm not only in pre-menopausal but also in post-menopausal patients, especially in the luminal B-like tumor subgroup.\u003c/p\u003e\n\u003cp\u003eIt is important to note that the RANK signaling has been identified as a crucial pathway in tumor initiation, inducing proliferation in normal epithelium and hyperplasia, but not in advanced lesions (ductal carcinoma in situ and adenocarcinomas) (2). These data are consistent with negative clinical results from D-BIOMARK and D-BEYOND regarding Ki67 and cleaved caspase-3. Once the tumor is established, the anti-proliferative and pro-apoptotic effect may be lost; however, there must be a mechanism, yet unclear, for those tumors with pharmacological or genetic inhibition of the pathway in preclinical studies to have fewer metastases (11,12). This therapeutic effect has also been reflected in clinical practice, with the large adjuvant trial ABCSG-18 demonstrating the benefit of adding denosumab in progression-free survival, bone metastasis-free survival, and overall survival, a trial designed exclusively in the luminal population (23). This benefit may be related to an anti-tumoral immune activation generated by denosumab, a hypothesis to be considered following our results.\u003c/p\u003e\n\u003cp\u003eD-BIOMARK trial demonstrated the role of denosumab as an immune enhancer, evidenced by the increase of TILs found after treatment. Despite in the control group no significant increase in TILs was observed, we cannot discard that the inflammatory effect generated by the biopsy, or differences between the biopsy and surgery specimens may contribute to increase TILs, as both control and experimental arm showed the same trend of elevated TILs. However, it is important to note that also in the analyses by subgroups, the increase in TILs only reached significance in the denosumab-treated patients, including pre and postmenopausal, and luminal B-like tumors despite the lower number of samples. Although the increase in TILs is modest to have clinical relevance, it should be noted that patients only received \u0026nbsp;2 doses of denosumab with one week of interval, and the analysis by the different subgroups was consistent with the increase in TILs reported in the experimental arm.\u003c/p\u003e\n\u003cp\u003eAn important implication of these results is the potential use of denosumab as an enhancer of immune infiltration in neoadjuvant therapy for luminal B-like tumors, where immunotherapy with pembrolizumab (KEYNOTE-756 clinical trial) or nivolumab (CheckMate 7FL clinical trial) has shown to improve pCR but with rates close to 25%, much lower than in TNBC (39,40). Could denosumab improve these results? During neoadjuvant chemotherapy, initially elevated levels of TILs correlate positively with a higher rate of achieving a pCR in all breast cancer subtypes (41). A booster given by denosumab allowing new combinations of treatments in a tumor microenvironment that would otherwise be cold is of clinical interest. The GeparX study already combined denosumab with neoadjuvant chemotherapy without reporting additional benefits. Although an immediate impact on pCR may not be evident, the possibility of a long-term effect cannot be ruled out (25\u0026ndash;28), and perhaps the best partner is the immunotherapy. There are already preclinical data showing synergy between immune checkpoint inhibitors (ICIs) and inhibitors of RANK signaling in solid tumors (29). The safety of the combination in clinical practice was reported in the CHARLI trial, a phase I/II study of the effect of denosumab with nivolumab (an anti-PD-1), with or without ipilimumab (anti-CTLA4), in patients with metastatic melanoma, which showed that the combination is safe and with at least interesting response rates (42). Pembrolizumab and denosumab have also been tested in clear cell renal cell carcinoma, a phase II trial (KeyPAD trial) with response rates close to 31% (43), and there are other ongoing trials with ICI in different tumors such as in lung cancer (Popcorn Trial) (44). Testing the combination in BC will be of interest, although we recognize that more data are required.\u003c/p\u003e\n\u003cp\u003eAdditionally, we have reported the expression of RANK and RANKL in tumor cells and stroma. A total of 34.54% of cases expressed RANK and 30% RANKL in the core biopsy, defined as an H-score \u0026gt; 0, slightly higher than reported in the literature, particularly in luminal tumors (18,19). In line with prior studies, RANK expression determined by IHC in tumor cells was associated with ER-negative tumors and high proliferative capacity (17\u0026ndash;19). Our data confirm that RANK protein expression in tumor cells could serve as a biomarker for tumor aggressiveness: a correlation was observed between high Ki67, low ER expression, and high histological grade. Recent data from the GerpaX clinical trial showed that RANK expression in tumor cells was an independent predictive biomarker of response to neoadjuvant chemotherapy in luminal breast cancer, highlighting the opposite in TNBC and HER2-positive tumors (28). Apparently, and according to our data, luminal tumors might also have the RANK pathway involved in their pathophysiology, not only TNBC, where the biological effect of RANK has been reported (18,19,45).\u003c/p\u003e\n\u003cp\u003eStrikingly our data reveals a novel association between RANK protein expression in the tumor microenvironment (stroma) with high levels of tumor proliferation (Ki67), which remains when studying exclusively luminal tumors (p=0.001). RANK expression in immune cells is predominantly found in myeloid cells such as macrophages and dendritic cells, while RANKL is predominantly found in TILs (20). Indeed, RANKL expression in the stroma of luminal tumors was associated with high percentage of TILs. Whether the highly proliferative tumors with RANK+ in the stroma have a greater immunosuppressive immune infiltrate is a hypothesis to be tested in the future.\u003c/p\u003e\n\u003cp\u003eWe were unable to establish a relationship between RANK or RANKL expression, neither in the stroma nor in the tumor cells, as a predictive marker for response to denosumab. However, RANK IHC in tumor cells was the only parameter in the multivariate analysis related to an increase of \u0026gt;10% in TILs between biopsy and surgery. This suggests that RANK expression in tumor cells may serve as a marker for aggressive tumors capable of recruiting higher levels of TILs. The difficulty in detecting RANK or RANKL as a response biomarker may be attributed to the variability and lack of standardization in the immunohistochemical technique for this staining, as well as the lack of a standardized cutoff point. In addition, the response, as evidenced by an increase in TILs after only two doses, was not strong enough to classify the change as a response, making it impossible to identify a biomarker.\u003c/p\u003e\n\u003cp\u003eRegarding serum markers, the reduction in sRANKL after denosumab administration unequivocally confirms the inhibition of the RANK pathway in our study. However, the unexpected absence of changes in bone remodeling markers TRACP5b and CTX may be attributed to the short time interval between both samples and/or to the kinetics of the markers, a finding that contrasts with the results of the D-BEYOND trial (29). Nevertheless, the reduction in serum calcium levels in the experimental group serves as a surrogate marker of the impact on bone remodeling in the experimental arm. Additionally, there was an increase in OPG in the experimental group, related to the decrease in sRANKL, suggesting an incremental feedback loop or that denosumab binding to RANKL \u0026quot;displaces\u0026quot; OPG, increasing the detection of free OPG in circulation. Our analysis revealed a significant negative correlation between both markers. Surprisingly, post-menopausal women in our study displayed higher OPG levels, diverging from what is reported in the literature (46). The complexity of interpreting biomarker dynamics is underscored by factors such as bone mineral density, body mass index, and cardiovascular disease, which were not collected at enrollment (47,48). Additionally, sRANKL levels showed no association with age or menopausal status. These nuanced findings emphasize the need for comprehensive data collection to unravel the multifaceted influences on this pathway.\u003c/p\u003e\n\u003cp\u003eAs limitations of our study, we can highlight the small sample size limiting subgroup analyses and the tumor heterogeneity, especially affecting the cohort of TNBC, with a clear selection bias since most of these tumors receive neoadjuvant chemotherapy. This subgroup only includes 5 out of 10 typical aggressive TNBC, 2 cases with low proliferation index tumors and 3 cases of apocrine neoplasms. As strengths of our proposal, we have a control group that prevents erroneous conclusions; in addition, the inclusion of a higher number of samples allowed us not only to confirm the denosumab driven increase in TILs observed on tumors from premenopausal women, but also to extend these findings to postmenopausal BC and luminal B tumors. This study provides clinical validation of preclinical observations and previous studies, placing us in a closer reality of the actual effect of denosumab in early breast cancer (49).\u003c/p\u003e\n\u003cp\u003eConsequently, our study serves as a crucial tool for understanding the behavior of this pathway within breast tumor cells, tumor microenvironment, and serum markers and opens new hypotheses for the implications of denosumab as a therapeutic target in BC, beyond its bone-related effects. This knowledge is essential for developing new drugs and for the more efficient utilization of denosumab.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eTwo doses of denosumab before surgery did not reduce tumor proliferation or increase tumor apoptosis. However, this short course of denosumab increased TILs in early BC, particularly in luminal B-like tumors, and in pre-and postmenopausal BC. These findings suggest that denosumab may enhance the body\u0026apos;s immune response against breast cancer, paving the way for further exploration and treatment refinement. Moreover, this trial underscores the complex nature of the RANK pathway, highlighting the necessity for further investigation into its multifaceted aspects.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e It\u0026rsquo;s an Investigator Sponsored Study: Amgen has assisted in the funding. The findings expressed herein are solely those of the authors, and Amgen does not assume any responsibility therefor. The study was sponsored by Institut Catal\u0026agrave; d\u0026apos;Oncologia. Additionally, grants were provided by the European Research Council (ERC Consolidator No. 682935 to E. Gonzalez), the Ministry of Science, Innovation, and Universities (Juan de la Cierva contract IJCI-2017-31564 to EM Trinidad), the Carlos III Health Institute (Rio Hortega contract CM19/00148 to A Vethencourt), and the Spanish Breast Cancer Research Group GEICAM (Balil-Pelegr\u0026igrave; grant to A Vethencourt).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement:\u0026nbsp;\u003c/strong\u003eThe study was conducted in accordance with the Declaration of Helsinki, and approved by the Institutional Ethics Committee of the \u0026ldquo;Institut Catal\u0026agrave; d\u0026rsquo;Oncologia de L\u0026rsquo;Hospitalet (ICO-L\u0026rsquo;Hospitalet)\u0026rdquo; (protocol code\u0026nbsp;\u003cstrong\u003ePR035/21\u003c/strong\u003e)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent Statement:\u0026nbsp;\u003c/strong\u003eInformed consent was obtained from all subjects involved in the study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e We extend our heartfelt gratitude to all team members involved in the project, encompassing the medical oncology, radiology, pathology, and surgery teams. Special recognition is extended to Ariadna Iserte (ICO) for her exceptional coordination, and to Maria Dolores Mulero and the histology team at Idibell for their invaluable assistance with staining procedures. We also thank Emilia Brizzi and Marta Matas for their help with image interpretation and statistical analysis. Additionally, our appreciation extends to Cristina Moreno, Valentin Navarro and all individuals who contributed in various capacities. Finally, we are deeply grateful to the patients and their families who generously participated in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u003c/strong\u003e No conflicts of interest were reported.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Materials:\u0026nbsp;\u003c/strong\u003eThe following supporting information can be downloaded at \u0026hellip;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAnderson DM, Maraskovsky E, Billingsley WL, Dougall WC, Tometsko ME, Roux ER, et al. A homologue of the TNF receptor and its ligand enhance T-cell growth and dendritic-cell function. Nature [Internet]. 1997 Nov 13 [cited 2021 Feb 25];390(6656):175\u0026ndash;9. Available from: https://pubmed.ncbi.nlm.nih.gov/9367155/\u003c/li\u003e\n\u003cli\u003eGonzalez-Suarez E, Jacob AP, Jones J, Miller R, Roudier-Meyer MP, Erwert R, et al. 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The statistics of phase 0 trials. 2010 [cited 2024 Mar 16]; Available from: www.interscience.wiley.com\u003c/li\u003e\n\u003cli\u003eCardoso F, McArthur HL, Schmid P, Cort\u0026eacute;s J, Harbeck N, Telli ML, et al. LBA21 KEYNOTE-756: Phase III study of neoadjuvant pembrolizumab (pembro) or placebo (pbo) + chemotherapy (chemo), followed by adjuvant pembro or pbo + endocrine therapy (ET) for early-stage high-risk ER+/HER2\u0026ndash; breast cancer. Ann Oncol [Internet]. 2023;34:S1260\u0026ndash;1. Available from: http://dx.doi.org/10.1016/j.annonc.2023.10.011\u003c/li\u003e\n\u003cli\u003eLoi S, Curigliano G, Salgado RF, Romero Diaz RI, Delaloge S, Rojas C, et al. LBA20 A randomized, double-blind trial of nivolumab (NIVO) vs placebo (PBO) with neoadjuvant chemotherapy (NACT) followed by adjuvant endocrine therapy (ET) \u0026plusmn; NIVO in patients (pts) with high-risk, ER+ HER2\u0026minus; primary breast cancer (BC). Ann Oncol [Internet]. 2023 Oct 1 [cited 2024 Feb 25];34:S1259\u0026ndash;60. Available from: http://www.annalsofoncology.org/article/S0923753423041546/fulltext\u003c/li\u003e\n\u003cli\u003eDenkert C, von Minckwitz G, Darb-Esfahani S, Lederer B, Heppner BI, Weber KE, et al. Tumour-infiltrating lymphocytes and prognosis in different subtypes of breast cancer: a pooled analysis of 3771 patients treated with neoadjuvant therapy. Lancet Oncol [Internet]. 2018 Jan 1 [cited 2024 Feb 25];19(1):40\u0026ndash;50. Available from: http://www.thelancet.com/article/S147020451730904X/fulltext\u003c/li\u003e\n\u003cli\u003eLau PKH, Harris SJ, Eastgate MA, Kee D, Mant A, Nott LM, et al. CHARLI: A phase Ib/II trial of ipilimumab-nivolumab-denosumab or nivolumab-denosumab in patients with unresectable stage III and IV melanoma. https://doi.org/101200/JCO20234116_suppl9525 [Internet]. 2023 May 31 [cited 2024 Feb 22];41(16_suppl):9525\u0026ndash;9525. Available from: https://ascopubs.org/doi/10.1200/JCO.2023.41.16_suppl.9525\u003c/li\u003e\n\u003cli\u003eGedye C, Harris CA, Stockler MR, Morris M, Ferguson T, Goh JCH, et al. 1886P Pembrolizumab and denosumab in clear cell renal cell carcinoma (ccRCC): A phase II trial (KeyPAD, ANZUP1601). Ann Oncol [Internet]. 2023 Oct 1 [cited 2024 Feb 24];34:S1014. Available from: http://www.annalsofoncology.org/article/S0923753423019531/fulltext\u003c/li\u003e\n\u003cli\u003eAhern ES, Cubitt A, Ballard E, Teng MWL, Dougall WC, Smyth MJ, et al. Preoperative PD1 checkpoint blockade and receptor activator of NFkB ligand (RANKL) inhibition in non-small cell lung cancer (NSCLC) (POPCORN). https://doi.org/101200/JCO2019378_supplTPS129 [Internet]. 2019 Jul 11 [cited 2024 Feb 24];37(8_suppl):TPS129\u0026ndash;TPS129. Available from: https://ascopubs.org/doi/10.1200/JCO.2019.37.8_suppl.TPS129\u003c/li\u003e\n\u003cli\u003eGomes I, de Almeida BP, D\u0026acirc;maso S, Mansinho A, Correia I, Henriques S, et al. Expression of receptor activator of NFkB (RANK) drives stemness and resistance to therapy in ER+HER2- breast cancer. Oncotarget [Internet]. 2020 May 5 [cited 2024 Feb 25];11(19):1714. Available from: /pmc/articles/PMC7233807/\u003c/li\u003e\n\u003cli\u003eDavis SR, Lambrinoudaki I, Lumsden M, Mishra GD, Pal L, Rees M, et al. Menopause. Nat Rev Dis Prim 2015 11 [Internet]. 2015 Apr 23 [cited 2023 Aug 8];1(1):1\u0026ndash;19. Available from: https://www.nature.com/articles/nrdp20154\u003c/li\u003e\n\u003cli\u003eRogers A, Saleh G, Hannon RA, Greenfield D, Eastell R. Circulating Estradiol and Osteoprotegerin as Determinants of Bone Turnover and Bone Density in Postmenopausal Women. 2002 [cited 2023 Aug 8]; Available from: https://academic.oup.com/jcem/article/87/10/4470/2846388\u003c/li\u003e\n\u003cli\u003eTschiderer L, Willeit J, Schett G, Kiechl S, Willeit P. Osteoprotegerin concentration and risk of cardiovascular outcomes in nine general population studies: Literature-based meta-analysis involving 26,442 participants. PLoS One [Internet]. 2017 Aug 1 [cited 2023 Aug 8];12(8). Available from: /pmc/articles/PMC5570489/\u003c/li\u003e\n\u003cli\u003eCiscar M, Trinidad EM, Perez-Montoyo H, Alsaleem M, Jimenez-Santos MJ, Toss M, et al. RANK is an independent biomarker of poor prognosis in estrogen receptor-negative breast cancer and a therapeutic target in patient-derived xenografts. bioRxiv [Internet]. 2021 Dec 14 [cited 2022 Mar 25];2021.12.13.470911. Available from: https://www.biorxiv.org/content/10.1101/2021.12.13.470911v1\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 6 are available in the Supplementary Files section.\u003c/p\u003e\n\u003cp\u003eTable 7 is not available with this version.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"breast-cancer-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"brcr","sideBox":"Learn more about [Breast Cancer Research](http://breast-cancer-research.biomedcentral.com)","snPcode":"13058","submissionUrl":"https://submission.nature.com/new-submission/13058/3","title":"Breast Cancer Research","twitterHandle":"@BCRJournal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"breast cancer, RANK, RANKL, denosumab, TILs, immune enhancer, tumor cell proliferation, tumor cell survival, HER2-negative","lastPublishedDoi":"10.21203/rs.3.rs-4283385/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4283385/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e The RANK pathway has been extensively investigated for its role in bone resorption; however, its significance extends beyond bone metabolism. Preclinical models suggest that inhibition of RANK signaling can prevent mammary tumor development by reducing proliferation and tumor cell survival. Additionally, both preclinical and clinical data support the ability of RANK pathway inhibitors to enhance the anti-tumor immune response.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e D-BIOMARK is a prospective, randomized window-of-opportunity clinical trial assessing the biological effects of denosumab, a monoclonal antibody against RANKL, in patients with HER2-negative early breast cancer. The study aims to assess denosumab's impact on breast tumor cell proliferation, apoptosis, and its potential to influence the tumor immune microenvironment. A total of 60 patients were enrolled and randomized 2:1 to receive two doses of single agent denosumab (120 mg one week apart) before surgery or to the control arm (no treatment). Fifty-eight patients were evaluated, 27 pre-menopausal, and 31 post-menopausal women, 48 with luminal tumors and 10 with triple negative breast cancer. Paired tumor samples were collected to compare baseline (core biopsy) and surgical (surgical specimen) time points, as well as serum samples at both time points.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Denosumab demonstrated its ability to reduce serum free RANKL levels (experimental p\u0026lt;0.001, control p=0.270). However, a reduction in tumor cell proliferation or cell survival was not observed. A denosumab-driven increase in tumor infiltrating lymphocytes (TILs) was observed (experimental p=0.001, control p=0.060), particularly in the luminal B-like population (experimental p=0.012, control p=0.070) and a similar trend in the TNBC group (experimental p=0.079, control p=0.237). Denosumab led to increased TILs in both pre-menopausal (experimental p=0.048, control p=0.639) and post-menopausal (experimental p=0.041, control p=0.062) women with luminal tumors. RANK protein expression in tumor and stroma was associated with markers of tumor aggressiveness but an increase in TILs was observed in the experimental arm irrespectively of RANK and RANKL expression in tumor or stromal cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e The D-BIOMARK trial highlights the potential of denosumab as an immune-enhancing agent in early HER2-negative breast cancer. Although preoperative denosumab did not reduced tumor proliferation or increased apoptosis, it led to an increase in TILs, particularly in luminal B-like tumors. These findings underscore the importance of further investigation into the multifaceted aspects of the RANK pathway.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTrial registration: \u003c/strong\u003eEudraCT number: 2016-002678-11 registered on June 15, 2018.\u003c/p\u003e\n\u003cp\u003eClinicalTrials.gov identifier: NCT03691311, retrospectively registered on September 04, 2018.\u003c/p\u003e","manuscriptTitle":"Denosumab as an immune modulator in HER2-negative early breast cancer: results of the window-of-opportunity D-BIOMARK clinical trial.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-29 16:34:02","doi":"10.21203/rs.3.rs-4283385/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-01-12T22:05:34+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-12T10:47:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"320994774501038771252046951403461287470","date":"2024-09-30T06:43:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"90190065029097021998505934261799943526","date":"2024-09-27T15:55:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"312913446949385327575383395212477473836","date":"2024-07-11T12:02:22+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-17T21:20:03+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-22T13:50:29+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-04-22T13:07:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"Breast Cancer Research","date":"2024-04-17T17:22:07+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"breast-cancer-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"brcr","sideBox":"Learn more about [Breast Cancer Research](http://breast-cancer-research.biomedcentral.com)","snPcode":"13058","submissionUrl":"https://submission.nature.com/new-submission/13058/3","title":"Breast Cancer Research","twitterHandle":"@BCRJournal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1dd271f3-2707-41f5-aeb9-cba38d3a4d85","owner":[],"postedDate":"April 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-05-19T16:06:57+00:00","versionOfRecord":{"articleIdentity":"rs-4283385","link":"https://doi.org/10.1186/s13058-025-01996-w","journal":{"identity":"breast-cancer-research","isVorOnly":false,"title":"Breast Cancer Research"},"publishedOn":"2025-05-12 15:57:56","publishedOnDateReadable":"May 12th, 2025"},"versionCreatedAt":"2024-04-29 16:34:02","video":"","vorDoi":"10.1186/s13058-025-01996-w","vorDoiUrl":"https://doi.org/10.1186/s13058-025-01996-w","workflowStages":[]},"version":"v1","identity":"rs-4283385","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4283385","identity":"rs-4283385","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}