Introduction: Neoadjuvant chemotherapy in breast cancer offers the possibility to facilitate breast and axillary surgery; it is a test of chemosensibility in vivo with significant prognostic value and may be used to tailor adjuvant treatment according to the response. Material and Methods: A retrospective single-institution cohort of 482 stage II and III breast cancer patients treated with neoadjuvant chemotherapy based on anthracycline and taxans, plus antiHEr2 in Her2-positive cases, was studied. Survival was calculated at 5 and 10 years. Kaplan–Meier curves with a log-rank test were calculated for differences according to age, BRCA status, menopausal status, TNM, pathological and molecular surrogate subtype, 20% TIL cut-off, surgical procedure, response to chemotherapy and the presence of vascular invasion. Results: The pCR rate was 25.3% and was greater in HER2 (51.3%) and TNBC (31.7%) and in BRCA carriers (41.9%). The factors independently related to patient survival were pathology and molecular surrogate subtype, type of surgery, response to NACT and vascular invasion. BRCA status was a protective prognostic factor without reaching statistical significance, with an HR 0.5 (95%CI 0.1–1.4). Mastectomy presented a double risk of distant recurrence compared to breast-conservative surgery (BCS), supporting BCS as a safe option after NACT. After a mean follow-up of 126 (SD 43) months, luminal tumors presented a substantial difference in survival rates calculated at 5 or 10 years (81.2% compared to 74.7%), whereas that for TNBC was 75.3 and 73.5, respectively. The greatest difference was seen according to the response in patients with pCR, who exhibited a 10 years DDFS of 95.5% vs. 72.4% for those patients without pCR, p < 0001. This difference was especially meaningful in TNBC: the 10 years DDFS according to an RCB of 0 to 3 was 100%, 80.6%, 69% and 49.2%, respectively, p < 0001. Patients with a particularly poor prognosis were those with lobular carcinomas, with a 10 years DDFS of 42.9% vs. 79.7% for ductal carcinomas, p = 0.001, and patients with vascular invasion at the surgical specimen, with a 10 years DDFS of 59.2% vs. 83.6% for those patients without vascular invasion, p < 0.001. Remarkably, BRCA carriers presented a longer survival, with an estimated 10 years DDFS of 89.6% vs. 77.2% for non-carriers, p = 0.054. Conclusions: Long-term outcomes after neoadjuvant chemotherapy can help patients and clinicians make well-informed decisions.

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 suggests a potential role for denosumab as an immune-enhancing agent in early HER2-negative breast cancer. Although preoperative denosumab did not reduce 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.

New breast cancer (BC) diagnoses will soon reach 2.5–3 million/year worldwide, with 15–25% of them being triple-negative breast cancer (TNBC), the most aggressive type, characterized for lacking the main pharmacological targets: estrogen and progesterone receptors (ERs and PRs), as well as HER2 overexpression. Therefore, chemotherapy remains the almost-unique systemic treatment for TNBC. However, some targeted therapies are recommended for use in combination with chemotherapy; namely, PARP inhibitors for BRCA-mutated TNBC, the immune checkpoint inhibitors pembrolizumab and atezolizumab, as well as the antibody–drug conjugates sacituzumab govitecan and trastuzumab deruxtecan, the latter for HER2low subtypes. Regardless of the limited benefits they provide, other treatments with similar mechanisms of action are being investigated in advanced clinical stages. Further, therapies that benefit other cancers, like PI3K/Akt/mTOR pathway and CDK4/6 inhibitors, are still being investigated for TNBC, although convincing results have not been obtained. Given this scenario, it might appear innovation for TNBC treatments has become stuck. However, the huge unmet medical need drives intense research into the biology of the disease. As a result, emerging disruptive therapies are being tested in early-stage trials, designed for novel targets and applying cutting-edge advances in immunotherapy and precision oncology.

Hormone receptor-positive, HER2-negative breast cancers are often poorly infiltrated by immune cells and derive limited benefit from current immunotherapy strategies. Here, using paired tumour samples from the randomised window-of-opportunity D-BIOMARK trial ( NCT03691311 ), we investigated the immunomodulatory effects of denosumab in early luminal breast cancer. Short-term preoperative denosumab reduced tumour-cell proliferative transcriptional programs and immunosuppressive features of the local tumour microenvironment, enhancing innate and adaptive immune activation and altering circulating cytokine profiles. High-resolution spatial transcriptomics revealed coordinated remodelling of tumour, immune, fibroblast and endothelial compartments after treatment. Denosumab reduced immune–tumour spatial separation and enhanced T cell activation, accompanied by a shift from matrix-associated tumour programs towards increased tumour–T cell communication. Copy number-informed tumour-state inference further identified a reduced representation of genomically complex, immune-poor tumour subclones after treatment. Together, these findings identify denosumab as a modulator of tumour–microenvironment crosstalk and support RANKL blockade as a strategy to render immune-poor luminal breast tumours more permissive to immune engagement.

Introduction: Neoadjuvant chemotherapy in breast cancer offers the possibility to facilitate breast and axillary surgery, it is a test of chemosensibility in vivo with significant prognostic value and may tailor adjuvant treatment according to response. Material and Methods: a retrospective single institution of 482 stage II and III breast cancer patients treated with neoadjuvant chemotherapy based on anthracycline and taxans plus antiHEr2 in those Her2 positive cases. Survival was calculated at 5 and 10 years. Kaplan Meier with log rank test were calculated for difference according to TNM, pathological and molecular surrogated subtype, TILs cut-off 20%, response to chemotherapy and the presence of vascular invasion. Results: In the univariate those factors that influenced long-term patient outcome were TNM (p=0.000); pathologic subtype (p=0.003); Tils cut off 20%; achieving pCR (p=0.000), RCB (p=0.000) and vascular invasion (p=0.000). After a mean follow up of 126 months, Luminal tumors presented a substantial difference of survival rates calculated at 5 or 10 years (81.2% compared to 74.7%) whereas for TNBC was 75.3 and 73.5, respectively. The greatest difference was seen according to response those patients with pCR presented a 10yDDFS of 95.5 vs 72.4% for those patients without pCR. This difference was especially meaningful in TNBC were 10y DDFS according to the RCB 0 to 3 was 100%, 80.6%, 69% and 49.2% respectively. Patients with a special bad prognosis in our series were lobular carcinomas with 10y DDFS of 42.9% vs 79.7%, p= 0.001 for ductal carcinomas; and those patients with vascular invasion at the surgical specimen with a 10yDDFS of 59.2% vs 83.6, p=0.,000 for those patients without vascular invasion. Conclusion: long-term outcomes after neoadjuvant chemotherapy can help patient and clinician make well-informed decisions.